Stapled Peptides Inhibitors: a New Window for Target Drug Discovery

CORE Metadata, citation and similar papers at core.ac.uk

Provided by University of Groningen

University of Groningen

Stapled Peptides Inhibitors Ali, Ameena M; Atmaj, Jack; Van Oosterwijk, Niels; Groves, Matthew R; Dömling, Alexander

Published in: Computational and Structural Biotechnology Journal

DOI: 10.1016/j.csbj.2019.01.012

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record

Publication date: 2019

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA): Ali, A. M., Atmaj, J., Van Oosterwijk, N., Groves, M. R., & Dömling, A. (2019). Stapled Peptides Inhibitors: A New Window for Target Drug Discovery. Computational and Structural Biotechnology Journal, 17, 263-281. https://doi.org/10.1016/j.csbj.2019.01.012

Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

Download date: 13-11-2019 Computational and Structural Biotechnology Journal 17 (2019) 263–281

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/csbj

Mini Review Stapled Peptides Inhibitors: A New Window for Target Drug Discovery

Ameena M. Ali, Jack Atmaj, Niels Van Oosterwijk, Matthew R. Groves, Alexander Dömling ⁎

Department of Drug Design, University of Groningen, Antonius Deusinglaan1, 9700AD Groningen, the Netherlands article info abstract

Article history: Protein-protein interaction (PPI) is a hot topic in clinical research as protein networking has a major impact in Received 22 November 2018 human disease. Such PPIs are potential drugs targets, leading to the need to inhibit/block specific PPIs. While Received in revised form 28 January 2019 small molecule inhibitors have had some success and reached clinical trials, they have generally failed to address Accepted 29 January 2019 the flat and large nature of PPI surfaces. As a result, larger biologics were developed for PPI surfaces and they have Available online 19 February 2019 successfully targeted PPIs located outside the cell. However, biologics have low bioavailability and cannot reach α Keywords: intracellular targets. A novel class -hydrocarbon-stapled -helical peptides that are synthetic mini-proteins fi Stapled peptide locked into their bioactive structure through site-speci c introduction of a chemical linker- has shown promise. PPI Stapled peptides show an ability to inhibit intracellular PPIs that previously have been intractable with traditional Drug discovery small molecule or biologics, suggesting that they offer a novel therapeutic modality. In this review, we highlight Inhibitor what stapling adds to natural-mimicking peptides, describe the revolution of synthetic chemistry techniques and Synthetic chemistry how current drug discovery approaches have been adapted to stabilize active peptide conformations, including ring-closing metathesis (RCM), lactamisation, cycloadditions and reversible reactions. We provide an overview on the available stapled peptide high-resolution structures in the protein data bank, with four selected structures discussed in details due to remarkable interactions of their staple with the target surface. We believe that stapled peptides are promising drug candidates and open the doors for peptide therapeutics to reach currently “undruggable” space. © 2019 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction...... 263 2. WhyStapledPeptides?...... 264 3. ChemicalSynthesisofStapledPeptides...... 265 3.1. Ring-ClosingMetathesis(RCM)...... 267 3.2. Lactamisation...... 268 3.3. Cycloadditions...... 269 3.4. ReversibleReactions...... 269 3.5. ThioetherFormation...... 269 4. StructuralInsightofStapledPeptidesTargetProtein-ProteinInteraction(PPI)inthePDB...... 270 4.1. SAH-p53-8:Stapledp53PeptideBindsPotentlytoHumanMDM2...... 270 4.2. MDM2DoubleMacrocyclizationStapledPeptide:FastSelectionofCell-ActiveInhibitor...... 270 4.3. SpeciﬁcMCL-1StapledPeptideInhibitorasApoptosisSensitizerinCancerCells...... 272 4.4. Stapled Peptides SP1, SP2 Inhibit Estrogen Receptor ERβ ...... 272 5. ComputationalApproachforStaplePeptideDesign...... 275 6. Conclusion...... 276 Acknowledgment...... 278 References...... 279

⁎ Corresponding author. E-mail address: [email protected] (A. Dömling). https://doi.org/10.1016/j.csbj.2019.01.012 2001-0370/© 2019 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 264 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

1. Introduction chemistry behind peptide stapling and provide an overview on some selected successful examples of peptide-based drugs to underline their Drug discovery approaches targeting protein-protein interactions importance. Lastly, we will underline four exclusive stapled-peptides (PPIs) has been fast-tracked over the present decade to deliver success- targeting PPIs, in which their staple makes an intimate interaction ful new drug leads and opens an expansive range of new therapeutic with the target interface, in order to reveal the role of stapling on pep- targets that were previously considered “undruggable”. This accelera- tide binding and their inhibition of PPIs. tion in PPI-based drugs is due to improved screening and design technologies, shortening the time between drug discovery to drug reg- 2. Why Stapled Peptides? istration and changing pharmaceutical economic delivery [1]. More- over, most human diseases are underpinned by a complex network of Helical peptides are one of the two main secondary structural ele- PPIs, (for example hubs such as p53), which underscores the need to ments in PPI interfaces, (in addition to β-sheets) and play a central understand PPIs not only on a clinical level, but also on molecular role in protein function within the cell. Often these elements are not sta- level. In this respect, the “omics” such as, genomics, RNA, proteomics ble in conformation in the absence of a complete protein fold. Addition- and metabolomics can accumulate huge volumes of data aiming at ally, peptides are sensitive to proteolysis by peptidases reducing their targeted and personalized medicine [2,3]. half-life (down to minutes), impacting their ability to penetrate cell All of the data, in addition to structural and screening-based membranes – all of which makes native peptides poor drug candidates approaches, have significantly expanded our understanding on PPI in- [14–16]. Notwithstanding, one main feature that makes peptides good terfaces that were previously highly challenging and difficult to target, drug candidates is their ability to bind large and relatively flat target sur- as these interacting surfaces are shallow or flat, non-hydrophobic and faces efficiently and specifically, which is a requirement in the majority large (1500-3000 Å). In addition, PPI surfaces differ in their shape and of intracellular therapeutically relevant PPIs. This makes peptides as an amino acid residue composition, particularly the hot spots that are es- attractive target for drug development and enables their transition into sential during binding protein partners; making small-molecules enti- the clinic [5,17]. The use of therapeutic peptides has grown explosively ties unlikely as protein therapeutics [4–8]. Moreover, the discovery of over the last three decades, covering areas such as metabolic diseases, innovative and drug lead molecules with the expected biological oncology, and cardiovascular diseases [ 18]. From a dataset that was col- activity and pharmacokinetics is the main aim of medicinal chemistry. lected recently on March 2018 and based on previously released data- Therefore, the application of ‘follow-on’-based strategy has always base report by Peptide Therapeutics Foundation, of 484 therapeutic been one of the most effective approaches that lead to promising bioac- peptides, 60 have been approved in the United States, Europe, and/or tive molecules. Conformational restrictions or “rigidification” is one of Japan, 155 peptides are in clinical development and 50% are currently these strategies that has been widely used to overcome ligand flexibil- in Phase II studies (Fig. 2)[18]. ity, which suffer from entropic penalty upon binding to the target sur- Massive efforts and optimizations have been conducted in order to face [9]. The restriction strategy has two major advantages: firstly, it overcome the limitations above. To impose a peptide α-helix conforma- could increase the potency of the drug-like agent by stabilizing a favor- tion (thereby improving their binding affinity toward their target pro- able binding conformation, reducing the entropic penalty on binding to tein) non-native amino acids were used in the peptide that lie on the the target and decrease its degradation by hindering metabolically labile same helix face. These non native amino acids are then linked together sites or introducing a fused-ring structure; in addition to improve iso- or “stapled” through side-chains that can be covalently bonded [16,19]. form selectivity or specificity toward targets. Secondly, controlling li- In order to address a second issue, to synthesize peptides with resis- gand confirmation could improve affinity on the atomic level without tance toward proteases, non-peptide (such as cyclic tripeptides, hetero- requiring additional interactions [9,10]. cyclic or other organic constraints) are inserted into a peptide sequence There are two types of drugs generally available on the market: tra- to maintain the peptide backbone in a linear saw-toothed strand struc- ditional small-molecule drugs with molecular weight of b500 Da and ture [20–23]. These chemical modifications have evolved over time high oral bioavailability but low target selectivity; and biologics that since the first all-hydrocarbon stapling by Verdine and colleagues in are typically N5000 Da (such as insulin, growth factors, erythropoietin 2000, who produced a large series of α, α-disubstituted non-natural (EPO) and engineered antibodies) that have limited oral bioavailability, amino acids bearing olefintethers(Fig. 3a). His work was an extension poor membrane permeability and metabolic instability. As a result such of Blackwell and Grubbs, who were the first to use Grubbs catalysts to medications are typically delivered by injection. However, biologics make a cross-link between O-allylserine residues on a peptide template have extremely high specificity and affinity for their targets due to the (Fig. 3b). Walensky provided the bridge between chemistry and biology large area of interaction with their targets [1,11]. Despite the success by generating hydrocarbon- stapled BH3 peptide helices, targeting BCL- of both drug classes in treating different diseases, there remains an op- 2 homology 3 domains responsible for the interactions of BCL-2 family portunity to offer a class of molecules to fill the gap in molecular weight proteins that mainly regulate cellular life and death at the mitochondrial between the existing two classes (Small molecules b500…Peptides…Bi- level. This stapled peptide not only showed a higher stability and re- ologics N5000 Da) and merge some of the advantages of small- markable resistance to proteolysis, but also high cellular permeability molecules and biologics in terms of oral bioavailability, cell penetration [19,24,25]. The details of stapled-peptide chemical synthesis will be and cheaper manufacturing costs. This class could be considered to be a discussed in detail in Section 3. next generation therapeutic class that precisely targets PPIs and is based Interestingly, peptides could be differentiated from proteins by their upon hydrocarbon-stapled α-helical peptides. Fig. 1 represents the size (50 amino acids or less) but have similar specificity toward their three classes of targeted drugs based on their molecular weight. targets as biologics. However, peptides are more potent binders to In this review we will focus on hydrocarbon-stapled α-helical pep- PPIs interfaces, because of their ability to bind large protein surfaces tides and their use as potential drugs. Hydrocarbon α-helical peptides with great selectivity and less toxicity when compared to small mole- are synthetic mini-proteins locked into their bioactive α-helix second- cule drugs, which often produce toxic metabolites. In contrast to small ary structure by site-specific insertion of a synthetic chemical staple molecules, peptides are degraded into amino acids, which are in turn linker or “brace”. Stapled peptides show a greatly improved pharmaco- not toxic or harmful for cells [1,26]. Furthermore, peptides have lower logic performance, increased affinity to their target, resistance to pro- manufacturing costs and are more stable at room temperature (unlike teolytic digestion, and afford high levels of cell penetration via recombinant antibodies and engineered proteins). Finally, as non- endocytic vesicle trafficking [5,12–14]. natural amino acids are the building blocks of peptides, the opportunity In this review we will discuss what stapling adds to this class of in- to produce diverse scaffolds with modified chemical and functional hibitors in terms of stability, bioactivity and cell penetration, the properties is available [27,28]. A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 265 Size Molecular Weight (Da)

<500 Da >5000 Da O CN N H

HO O S N O O H NH2 NH2 Me Ph CO2Me

Hydrocarbon Small-molecules α-helix peptides Biologics

Fig. 1. The three classes of targeted medicines. The traditional small- molecules inhibitors were the ﬁrst class discovered to inhibit different PPIs surfaces with MW of b500 Da and high bioavailability. Most of the biologics, the second class of PPI targeted molecules, have a MW of N5000 Da (eg. antibodies and growth hormones) aimed to overcome a broad range of diseases. Stapled α-helix peptides as a class address this gap in MW between small molecules and biologics, aiming to combine the oral bioavailability of small-molecules with the high speciﬁcity of biologics toward the target protein.

Structural knowledge of the target PPIs and mutagenesis data for structures are SAHBA, based on BH3 domain of proapoptotic BID protein residues at or near the binding interface are necessary to achieve a suc- [25], SAH-p53, based on the p53-MDM2 interaction interface [29], SAH- cessful interruption of PPI partner proteins in vivo. Peptide design gp41 double stapling peptide, targeting the HIV-1 virus and Enfuvirtide, is based upon the ligand-target pair, in that the ligand retains its the first decoy HR2 helix fusion inhibitor [30]. If the proteins involved in α-helical motif and is docked into shallow cleft surface of the target pro- the PPIs of interest have no previous structures, Ala-scanning or residue tein. Thus, stapled peptide inhibitors represent “dominant-negative” conservation “in situ mutagenesis” can be used as a starting point to po- versions of the docking helix [5]. The peptide is then optimized by se- sition the staple. If this information is also not available, then synthesiz- quence modification “or stapling” to improve cell penetration and pep- ing and screening all stapling positions is advisable [5]. tide efficacy to compete with the intracellular ligand protein, and it is crucial to position the cross-linking amino acids in such way that the 3. Chemical Synthesis of Stapled Peptides targeted interface remains intact [5]. After evaluating the cellular uptake of the stapled peptides using live confocal microscopy, a broad spectrum As the synthesis of bioactive-stapled peptides started to widen, the of cellular and in vivo studies are applied to examine the therapeutic ac- approaches used also branched and allowed stapled peptides to be tivity of the stapled peptides toward their targets. A flow-chart in Fig. 4 applied for various purposes such as target binding analyses, structure summarizes the development process of therapeutic peptides for bio- determination, proteomic discovery, signal transduction research, cellu- logical study, from virtual design to in vivo mouse model analysis. Exam- lar analyses, imaging, and in vivo bioactivity studies [31]. Solid-phase ples of stapled peptide created through the use of high-resolution peptide synthesis (SPPS) is a standard and commonly used chemical

Fig. 2. Statistical representation of therapeutic peptides until March 2017. The numbers are indicated in percentage at each category with a total number of 484 medicinal peptides that were produced with development activity regulatory approval from major pharmaceutical markets as, the United States, Europe, and Japan. From these peptides 12% were approved, while 32% are in clinical trials and further classiﬁed as phases I, II, III and pre-registered. The highest percentage (54%; “Discontinued”) category encompasses peptides terminated before approval. The lowest percentage 2% is the “Withdraw” category that refers to previously approved peptides that are no longer available in the market [18]. 266 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

a) α,α-disubstitution, Olefin tether (RCM)

H C CH 3 3 CH3 H3C

b) O-allyl Serine Residues (RCM)

O O O O

Fig. 3. Ruthenium-catalyzed ring-closing metathesis (RCM) reaction for peptides stapling was a) published for the first time by Verdine and Schafmeister in 2000 by engaging α,α- disubstituted non-natural amino acids harboring all-hydrocarbon tethers [19]. Their work was a continuation of b) Blackwell and Grubbs work in 1998 [24]; who performed ruthenium-catalyzed olefin metathesis for macrocyclisation of synthetic peptides using a pair of O-allylserine residues in a metathesis reaction. procedure to synthesize α-helix peptides. The first required entity to SPPS has been automated using Fmoc chemistry to become an start stapled peptides synthesis is a stock of non-natural amino acids efficient and reliable method to yield hydrocarbon-stapled peptides of building blocks with a variable length of the terminal olefin tethers. single or double stapling with different functionalities and experimental The choice of the non-natural amino acids will define the length, struc- applications. However, SPPS has two main complications: First, ture and the chemical functionalities of the stapled linker [14,32]. efficiency is limited in longer peptides (N50 residues). These are more The helix backbone amino acids are protected with a base-labile usually expressed using recombinant DNA technology, due to the un- fluorenylmethoxycarbonyl (Fmoc) to obtain N- α -Fmoc-protected availability of the N-terminal amine of the non-natural amino acids amino acids, which are often offered with acid-labile side chain (mostly after naturally bulky residues like arginine or β- branched protecting groups that vary between the 20 amino acids. The side amino acids (valine, isoleucine, and threonine)). Additionally, extension chain protecting groups of each amino acid for standard SPPS of stapled of deprotection and coupling times with fresh reagent may be required peptides are indicated in Table 1. After the synthesis of non-natural in the synthesis of larger peptides. The second complication is that amino acids and peptide elongation during SPPS; ring-closing metathe- cross-reaction or progressive inaccessibility of the N-terminus due to sis (RCM) of the stapled is performed. on-resin aggregation could occur [31–33].

Fig. 4. Workﬂow of all hydrocarbon-stapled peptides generated for biological investigation. Computational designation of the peptides including in-situ mutagenesis to screen all possibilities based on previous reported structures, followed by in vitro biochemical, structural, and functional studies compromising peptides binding afﬁnities measurements toward the target protein interface utilizing biophysical assays and crystallization trials. Potent binder peptides will be further tested for their cellular uptake and permeability using live confocal microscopy. Lastly, successful peptides are subjected to a broad spectrum of cellular and in vivo analyses, using mouse models of the studied disease. A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 267

Table 1 type and position. Stapling techniques could be divided into The acid-labile side chain protecting groups used in SPPS synthesis of stapled peptides. one-component or two-component stapling techniques, based on the Amino acid Three letters-code Side chain protecting group side-chain linking reaction. During one-component stapling a direct bond will be formed between two non-natural amino acids side- Alanine Ala N/A Cysteine Cys Trityl (Trt) chains, whereas two-component stapling involves a separate bifunc- Aspartic acid Asp tert-Butyl (OtBu) tional linker to connect the side-chains of two non-natural amino Glutamic acid Glu tert-Butyl (OtBu) acids [14]. The most commonly used technique for stapling is the one- Phenylalanine Phe N/A component stapling technique - employing S-pentenylalanine at i,i + Glycine Gly N/A Histidine His Trityl (Trt) 4 positions for one turn stapling or combining either R-octenylalanine/ Isoleucine Ile N/A S-pentenylalanine or S-octenylalanine/R-pentenylalanine at i,i + 7 po- Lysine Lys tert-Butoxy (Boc) sitions. Other spacings for stapling were also accomplished upon chem- Leucine Leu N/A ical optimization, including i,i + 3 and i,i + 11 [14,31,32,34]. The Methionine Met N/A common stapling positions are shown in Fig. 5. Asparagine Asn Trityl (Trt) Proline Pro N/A There are several chemical procedures to enclose or stabilized Glutamine Gln Trityl (Trt) the all-hydrocarbon linker into α-helix peptide such as, ring-closing Arginine Arg Pentamethyldihydrobenzofurane (Pbf) metathesis, lactamisation, cycloadditions, reversible reactions and Serine Ser tert-Butyl (OtBu) thioether formation. A brief summary for each methodology and some Threonine Thr tert-Butyl (OtBu) Valine Val N/A literature examples is provided below. Tryptophan Trp tert-Butoxy (Boc) Tyrosine Try tert-Butyl (OtBu) 3.1. Ring-Closing Metathesis (RCM)

Blackwell and Grubb were the ﬁrst to apply alkene ring-closing me- Initial screening of different types of stapling is required if structural- tathesis as a peptide stapling method. They described solution-phase based knowledge is not available. As indicated previously in Section 2, metathesis, followed by hydrogenation of hydrophobic heptapeptides prediction software can suggest the peptide α-helix template, then a containing either O-allyl serine or homoserine residues with i,i + 4 group of constructs with differentially localized staples can be gener- spacing (Fig. 6)[24]. Their study emphasized the feasibility of metathe- ated to determine the optimal staple placement. However, if the target sis on helical peptide side-chain. Later in 2000, Schafmeister and his PPIs interface is structurally well characterized, this structural data can colleagues managed to conduct metathesis stapling using α,α-disubsti- be used for computational docking and designing of the desired tem- tuted amino acids carrying oleﬁnic side-chains of different lengths and plate peptide to generate a panel of peptides with diverse stapling stereo-chemistry on solid phase prior to peptide cleavage from resin,

a) i,i+4 i,i+3

i,i+7 i,i+11

b) R8 R5 S8 S5

O O O O

H2N (R) H2N (R) H2N (S) H2N (S) OH OH OH OH

Me Me Me Me

Fig. 5. a) The common stapling insertion positions for α-helix peptides. Combinations of two non-natural amino acids S5, R5, S8 and R8 are used for different positions of stapling the hydrocarbon linker. Employing S5/S5 at position i,i + 4 is the most common stapling position on the same face of helix turn. For i,i + 7 position, two combinations could be applied either S8/R5 or S5/R8. Synthetic chemistry evolved to introduced i,i + 3 and i,i + 11 as new possible positions for stapling in addition to double-stapling. b) The structures of the four designed amino acids used to introduce all-hydrocarbon staples into peptides. All possess an α-methyl group (Me) and an α-alkenyl group, but with opposite stereochemical conﬁguration and different length at the alkenyl chain. 268 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

( )3 ( )6 ( )3 ( )6

Ac-EWAE NH AAAKFL HN AHA Ac-EWAE NH AAAKFL HN AHA

Fig. 6. RCM or ring closing metathesis reaction for synthesis of the all-hydrocarbon stapled peptide reported by Schafmeister et al. 2000, which increase peptides helicity as found by circular dichroism (CD) [19].

O O NH NH 2 HO ( )4 Ac NH C ARA NH C Ac NH C ARA NH C O O O O

Fig. 7. A Lactamisation study that was conducted by Fairlie and co-workers on penta and hexa-peptides in order to optimize lactam stapling between Orn/Lys and Asp/Glu residues. It wasn't the first study for lactam optimization; however, the group was abled to systematically and quantitatively found the shortest peptide with retained helicity in water as judged by CD [39]. producing a large series of α, α-disubstituted non-natural amino 3.2. Lactamisation acids S5, R5, S8 and R8 bearing olefin tethers that were used for different stapling positions of the hydrocarbon linker as shown in Fig. 5. Stabilization of an α-helix can also be accomplished through side- The end products were a collection of i,i + 4/7 peptides and they chain intramolecular amide-bond formation between i,i + 4 spaced found that i,i + 7 stapled peptides have higher helicity and stability amine- and carboxy-side chain amino acids. Lactamisation was first over native and non-stapled peptides [19]. BID BH3 peptides that studied by Felix et al. in 1988 [37], in which they coupled Lys and Asp bind to BCL-2 family proteins are a successful product of metathesis residues side-chains in a growth hormone releasing factor short conge- stapling by Walensky et al. and they showed that the optimized sta- ner. Growth hormone helicity and activity were conserved post pled peptide has more stability than the native one, provoke apopto- macrocyclisation, which were measured by NMR and circular dichroism sis in leukemia cells, and inhibit the growth of human leukemia (CD) (both methods that can be used for analyzing the secondary struc- xenografts in mice [25]. p53-MDM2/MDMX dual inhibitor stapled ture of peptides and proteins in aqueous solution) [38]. Since then, nu- peptides were reported by Sawyer and co-workers, who provided merous studies applied lactamisation and amide linkage on different promising in vitro data for binding affinity, cellular activity and sup- chain length and positions, with the intention of generating a stable pression of human xenograft tumors in animal models [35]. These helix for different systems. For example, a lactam stapling optimization findings are the basis of p53 optimized stapled peptides that have study on penta/hexapeptides between Orn/Lys and Asp/Glu residues, enter clinical trials. carried out by Fairlie and co-workers (Fig. 7)[39], examined the Further, Verdine et.al introduced a unique form of multiple stapling, shortest possible peptide with α-helix reinforced structure in water. called stitches, in which two hydrocarbon staples immediately follow Subsequently, the Fairlie group applied their finding on different tar- one another. This technique requires the use of the amino acid bis- gets, including inhibition of respiratory syncytial virus with double pentenylglycine (B5) that forms a junction between the two staples lactam-stapled peptide in 2010 with improved antibacterial activity. and emerges from a common residue in the peptide. There are many Another target was the nociceptin hormone studied in the same year, possible combinations of stereochemistry and linker length in such a in which lactam-stapled peptide induced higher ERK phosphorylation system. Various stitch combinations were studied rigorously and two in mouse cells and thermal analgesia [22]. Norton and co-workers also systems, i,i + 4 + 4 (S5 + B5 + R5) and i,i + 4 + 7 (S5 + B5 + S8), examined several Asp/Lys lactam-stapling combinations at i,i + 4 posi- appeared the most effective for helix stabilization. A peptide with the tion on μ-conotoxin KIIIA, a natural peptide from mice that acts as a po- latter stitch construction was found to have superior helicity and cell tent analgesic by binding voltage-gated sodium channels (VGSCs), penetration compared with an i,i + 7 stapled analogue [36]. where they found that stapled peptides have different level of helicity Optimization and extensive development in hydrocarbon stapling and inhibitory activity on variable VGSC when examined in Xenopus approach allow stapling at i,i + 3/4/7 spacings. Regardless of the many laevis oocytes [40]. From a chemical perspective, lactam stapling is examples in literature of successful hydrocarbon stapling, there is no easier to obtain and incorporate due to proteogenic amino acids guarantee that stapling will enhance peptide stability, cell penetration when compared to other stapling techniques, which require non- and binding to the target. Extensive optimization is needed in order to proteogenic amino acids. A drawback is that an extra orthogonal discover a staple peptide with the desired features. protecting group is needed for selective deprotection of the amino

N N N

( )4 Ac-LSQEQLEHR NH C RSL NH C TLRDIQRMLF-NH2 Ac-LSQEQLEHR NH C RSL NH C TLRDIQRMLF-NH2 O O O O

Fig. 8. Optimized CuAAC-stapled peptide was successfully developed to inhibit the BCL9 oncogenic interaction. After screening different stapling length, Wang and co-workers concluded that ﬁve units of methylene was optimal stapled peptide for BLC9 inhibition [43]. A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 269

AcNH NHAc S S S S ( )4 ( )4

Ac-AAA NH C KAAAAK N C AAAKA-NH2 Ac-AAA NH C KAAAAK N C AAAKA-NH H H 2 O O O O

Fig. 9. Schultz and co-workers described an i,i + 7 stapling methodology using disulphide bridges between D and L amino acids bearing thiol-side chains. The amino acids were connected with acetamidomethyl (Acm) protecting groups, deprotected and then oxidised with iodine to give a disulphide stapled peptide. CD spectra of disulphide stapled peptides exhibited a high level of α-helicity in comparison to the Acm-protected precursors that were significantly less helical [45]. acid functionalities prior to lactamisation. Another limitation of this 3.4. Reversible Reactions technique should be mentioned, which is the lactamisation stapling of Lys and Asp residues. Stapling at these residues is only compatible Using disulphide bridges between two Cys residues as stapling with i,i + 4 spacing with longer linkages that required modified technique was first introduced by Schultz et al. at i,i + 7 positions. amino acids with longer side-chains. From a biological point of view, The disulfide bridge was formed between D and L-amino acids having and based on a large number of studies on peptide lactamisation, this thiol-side chain, followed by the addition of acetamidomethyl (Acm) stapling technique can create therapeutic peptides with superior bioac- protecting groups, protection and oxidization with iodine (Fig. 9). tivity. However, most of their targets are either extracellular or The helicity of disulfide-stapled peptides was higher when compared membrane-bound, suggesting that lactamisation stapling has no poten- with the Acm-protected precursors, as displayed in CD spectroscopy tial to improve cell penetration [14]. [45]. Although disulfide stapling was the earliest reported stapling technique, little was concluded due to the instability of the disulphide stapled peptides in reducing environments, which restrict their appli- 3.3. Cycloadditions cation in intracellular targets. However, stapling with oxime linkages [46] and two-component bis-lactam and bis-aryl stapling techniques Cu (I)-catalyzed azide–alkyne cycloaddition (CuAAC) or the “Click” [47,48] were found to be superior to the analogous disulphide sta- reaction is another mechanism of peptide stapling, it is also known as pling. Recently, Wang and Chou demonstrated the possibility of sta- biocompatible ligation technique [41]. The first research group who pling and macrocyclization using thiol-en between two Cys residues applied CuAAC to generate α-helix structures between i,i + 4 spacing an α, ω-diene in high yields (an unsaturated hydrocarbon containing within peptides were Chorev, D'Ursi and co-workers in 2010, based on two double bonds between carbon atoms), which allowed stapling of parathyroid hormone-related peptide [42]. Subsequently, many both expressed/unprotected and synthetic peptides. This group ap- groups used this type of stapling in order to determine the best linker plied their discovery to the p53-MDM2 PPI and successfully synthe- length, including Wang and co-workers, who found that five methy- sized stapled peptides with both i,i + 4 and i,i + 7 linkages, lene units were the optimum staple length to inhibit the oncogenic applying this method in the stapling of large peptides and proteins. BCL9-beta-catenin PPI (Fig. 8). A further significant result, reported Development in reversible stapling is slow, but efforts in applying by the same team, of the Click reaction was based on triazol- this method in biological dynamic covalent chemistry are under active position screening along a peptide targeting the same oncogenic pro- investigation. tein, beta-catenin, to generate a library of stapled peptides exhibiting different in vitro binding affinities and helicity [43]. Madden et al., used an unusual cycloaddition via UV-induction between tetrazoles 3.5. Thioether Formation and alkenes to hinder p53-MDM2/MDMX interaction. The stapling reaction took place between i,i + 4 by exposing unprotected linear pep- The reaction between Cys thiol and alpha-bromo amide groups has tides to UV irradiation in solution, which resulted in stapled peptides been developed as a protocol for peptide stapling by Brunel and Dawson displaying higher affinity toward MDM2/MDMX in a fluorescence po- [49]. This linkage was designed to mimic the ring size of previously re- larization assay (FP). However, these peptides were not cell perme- ported lactam staples, but a thioether link was hosted into gp41- able. This problem could be overcome by modifying a number of the peptide epitopes as an approach to establish an HIV vaccine. Successful peptide amino acids to Arg, whereby cellular uptake and moderate staples were created in both i,i + 3 and i,i + 4 linkages and a peptide p53 activity were achieved [44]. Generally, stapling with cycloaddition with i,i + 3 stapling (Fig. 10) demonstrated a higher helicity over chemistry shows a promising future, in that triazol- stapled amino unstapled and lactam-stapled peptides i,i + 4. Moreover, after optimiza- acids are accessible and CuAAC is well established. In the example of tion the stapled peptide bound to a gp41-specific antibody (4E10) more UV-induced reactions, the method is simple to apply but requires effectively than the uncyclised peptide [50]. These findings illustrate the extra analysis that might affect applicability in other biological efficiency of thioether stapling with shorter distance i.e. i,i + 3, while systems. suggesting that lactam staples are more suitable for i,i + 4 stapling.

O Br O SH HN S HN ( ) ( )3 3

H2N C WF N C ITNWLWKKKK-NH H2N C WF N C ITNWLWKKKK-NH2 H 2 H O O O O

Fig. 10. Thioether stapling method was reported by Brunel and Dawson in 2005. They demonstrated the reaction of Cys thiol and alpha-bromo amide groups to reportai,i+3thioether stapled peptide that inhibited HIV fusion using the gp41 epitopes as template for peptide synthesis [49]. 270 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

4. Structural Insight of Stapled Peptides Target Protein-Protein revealed an extended region of the helical peptide from residues 19–27 Interaction (PPI) in the PDB in the bound state that was not seen in other peptides with lower affinities toward MDM2. Moreover, the bound peptide induced minor The number of peptides entering clinical trials has increased over the changes in MDM2, specifically at the side chain of Met62 (which folds last 35 years, with an average peaking in 2011, when over 22 peptides/ away from the p53 binding pocket, to make space for the staple), year were successful in entering clinical development [18]. This evolved Val93 (which shifts inside the binding pocket) and the side chain of from technology maturation and advances in synthetic chemistry and Tyr100 that is found in a “closed” form. However, the α-helix peptide purification of peptides, in parallel with improvements in biophysical is located in the same position as the native helix of p53, orienting the and molecular pharmacological methods. However, there is a limited three residues critical for binding (Phe19, Trp23 and Leu26) in the cor- number of high-resolution structures of staple peptides in complex rect location (Fig. 11). Remarkably, the aliphatic staple intimately inter- with their targets in the protein database (RCSB www.rcsb.org), as of acts with the protein and is located directly over the Met50-Lys64 helix July 2018 [51]. There are 67 “stapled peptides” structures, of which 58 and contributes ca. 10% of the peptide-Mdm2 total surface contact are based on X-ray diffraction (83%) and 9 on NMR studies (17%). area. Additionally, the staple shields a H-bond between Trp23 and When limiting the analysis to Homo sapiens, our search found 43 struc- Leu54 from solvent competition (Fig. 12). Two novel features were tures, targeting a limited range of PPIs, of which the majority are of the discovered in the complex structure, first an extended hydrophobic in- p53-MDM2/MDMX interaction, the BCL-2 family (including the MCL-1 terface of the staple linker with Leu54, Phe55, Gly58, and Met62 of BH3 domain), estrogen receptor and human immunodeficiency virus Mdm2. The second feature is that the staple displaced a common type 1 (HIV-1). Other druggable interfaces of interest were kinases water molecule present in most MDM2 structures, which forms [52,53], insulin [54], tankyrase-2 [55], growth factor receptor-bound H-bonds with Gln59-N and Phe55-O. The later displacement likely en- protein 7 (Grb7) [56], the Fc portion of human IgG [57], eIF4E protein tropically stabilizes the complex during binding and contributes to

[58] and transducin-like enhancer (TLE) proteins [59]. MDM2 and its SAH-p53-8 tight binding as evidenced by an FP assay showing a Kd of homolog MDMX represent 18.6% of total X-ray structures in protein 55 nM. Lastly, stapling increases peptide helicity during binding in rela- data bank, which indicates their importance as the main negative regu- tion to that of native p53 - influencing residue Leu26, which plays an lators of p53 (“The Guardian of the genome”) since it behaves as a hub important role in MDM2 binding. Additionally, the researchers con- protein [60]. This escalation in stapled peptide drug discovery has cluded that the long stapling i,i + 7 enhanced helical conformation crossed over into the traditional focus upon endogenous human pep- and affinity as suggested by previous studies [19]. Subsequent to the tides to include a broader range of structures identified through medic- discovery of SAH-p53-8, several stapled peptides, such as sMTide-02 inal chemistry efforts. Not surprising, today over 150 stapled peptides [99] and ASTP-7041 [35], showed potent binding toward MDM2 with are in the active development of human clinical studies [18]. Kd values of 34.35 and 0.91 nM, respectively. Additionally, both peptides The analysis presented in Table 2 provides a list of the crystal struc- (in addition to VIP-84 (another stapled peptide targeting MDM2: p53)) tures belong to therapeutic stapled peptides, which mimic the native showed cellular permeability when tested using a nanoBRET (Biolumi- peptides in complex with their target protein interfaces. The table also nescence Resonance Excitation Transfer) live cell assay. Screening vari- gives an overview of the PDB structure code, name of stapled peptide ous lipid based formulations, the cellular uptake of VIP-84 was shown to and biophysical assays that are used to measure the binding dissociation be enhanced, as well as its biological activity, which was linked to vesic- constant (Kd) of the stapled peptide to the target protein. ular or endosomal escape of the stapled peptide through the cell mem- Not all staples interact with the target protein surface via commonly brane [100]. known chemical interactions; instead they can induce conformational changes to either the synthetic α-helix peptide or the target protein in- 4.2. MDM2 Double Macrocyclization Stapled Peptide: Fast Selection terface, specifically the amino acids residues involved in PPIs. These of Cell-Active Inhibitor changes stabilize and fix the helical peptide in a potent binding mode within the target interface. A limited number of stapled peptides have Following on from the SAH-p53-8 potent peptide inhibitor, Lau et al. different interactions with their intracellular targets, which contributes managed to synthesize a stapled peptide-E1 by applying a novel sta- to their high specificity, stability and makes these peptides promising pling technique [64]. This technique is based on double Cu-catalyzed target therapies for human diseases. Table 3 underlines the role of the azide–alkyne cycloaddition (CuAAC) and followed two-component stapled linker in binding to the target protein surface and indicates if strategies, in that the staple and α-helix peptide are separated before it is involved in any interaction with the target surface residues via cyclisation. This was combined with click chemistry to generate a pep- Van der Waals, hydrogen/disulfide bonds or π-π interactions. All of tide with variable functional staples. The team used the p53-MDM2 in- these interactions were inspected from the crystal structures of the sta- teraction as a model, since that target has been well investigated as an pled peptides in complex with the target protein surfaces. Examples of oncogenic therapy for cancers with overexpressed MDM2. For optimum these peptides will be discussed extensively in the next sections to high- inhibitor screening, and to ensure fast and easy selection for the best light the evolution of medicinal chemistry techniques. peptide, cyclisation was conducted in situ and directly in primary cells medium using a 96-well assay. This approach eliminated the extra puri- 4.1. SAH-p53-8: Stapled p53 Peptide Binds Potently to Human MDM2 fication step required in other two-component strategies and provided a first example of stapling within a biological environment. The first sta- p53, the main tumor suppressor, which is mainly negatively regu- pled peptide A1 was synthesized by linking diyne 1 to p53-derived lated by the E3 ubiquitin ligase MDM2. Although p53 is mutated or diazidopeptide A to produce A1 with 60% yield. Different peptide vari- inactivated in N50% of human cancers, the other 50% retain WT-p53. ants B-E were tested in situ to define a peptide with the highest p53 ac- Therefore, the p53: MDM2 PPIs is a promising and confirmed target tivation, showing that the E+1 stapled peptide was the most potent for drug discovery and cancer therapy. This can be accomplished by dis- activator within cells. The binding affinity of the E1 peptide was mea- covering a potent MDM2 binder in order to prevent its binding to p53 sured using two biophysical assays (FP and ITC) determining Kd values and thereby restore its biological function. In 2012, Baek and co- of 7.5 ± 0.7 and 12 ± 3 nM, respectively. The crystal structure of E1- workers were able to resolve a high-resolution structure of a stapled MDM2 (17–108, E69A/K70A) complex at 1.9 Å resolution elucidated peptide inhibitor in complex with MDM2 (SAH-p53-8 (PDB 3V3B)) the helical structure of E1 orienting the three hydrophobic key residues [62]. This peptide was synthesized following ring-closing olefin metath- (Phe19, Trp23 and Leu26) in the correct positioning for MDM2 binding esis (RCM) at i,i + 7 stapling positions between residues Asn20 and (PDB 5AFG), in a manner broadly similar to that of the p53 native pep- Leu26. As anticipated by molecular dynamics (MD), the crystal structure tide (Fig. 13a). The bis (triazolyl) staple was discovered in an anti A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 271

Table 2 List of structural-resolved stapled peptides in complex with PPI targets from RCSB-PDB.

Target PDB ID Binding Assay Peptide Kd (nM) Ref. X-ray NMR

Human MDM2/4 IYCR ITC p53-WT Residues (15–29) 600 [61]NA 3V3B FP SAH-p53-8 Stapled peptide 55 [62]NA ITC 12 4UMN FP M06 Stapled peptide 63 ± 17.8 [63]NA 5AFG FP E1 Stapled peptide 7.5 ± 0.7 [64]NA 4UE1 FP YS-1 Stapled peptide 9.9 ± 1.5 [65]NA 4UD7 FP YS-2 Stapled peptide 7.4 ± 1.5 [65]NA 5XXK FP M011 Stapled peptide 6.3 ± 2.9 [66]NA 5VK0 – PMI – [67]NA 5VK1 – PMI – [67]NA

MCL-1/BCL-2 3MK8 FP MCL-1 SAHBD Stapled peptide 10 ± 3 [68]NA 5C3F FP BID-MM Stapled peptide 153 ± 12 [69]NA SPR 107 ± 29 5C3G SPR BIM-MM Stapled peptide 460 ± 232 [69]NA 5W89 FP SAH-MS1-18 Stapled peptide 25 ± 7 [70]NA 5W8F FP SAH-MS1-14 Stapled peptide 80 ± 5 [70]NA 5WHI – BCL-1 Apo – [71]NA 5WHH Streptavidin pull-down D-NA-NOXA SAHB Stapled peptide – [71]NA Estrogen Receptor 2YJD SPR SP1 ERβ/1.99 μM [72]NA αER/674 2YJA SPR SP2 ERβ/632 [72]NA αER/352 5DXB SPR SRC2-SP1 530 [73]NA 5HYR SPR SRC2-SP2 42 [73]NA 5DX3 SPR SRC2-SP3 39 [73]NA 5DXE SPR SRC2-SP4 – [73]NA 5DXG SPR SRC2-SP5 – [73]NA 2LDA – SP2 – NA [72] 2LDC – SP1 – NA [72] 2LDD – SP6 ERβ/155 NA [72] αER/75 5WGD – SRC2-LP1 – [74]NA 5WGQ – SRC2-BCP1 – [74]NA Aurora-A 5LXM ITC Stapled TPX2 peptide 10 0.18 μM[73]NA Tankyrase-2 5BXO FP Cp4n2m3 0.6 ± 0.01 μM[55]NA 5BXU FP Cp4n4m5 2.8 ± 0.1 μM[55]NA Grb7 5D0J SPR G7-B4NS peptide 4.93 ± 0.03 μM[56]NA 5EEL SPR G7-B4 peptide 0.83 ± 0.006 μM[56]NA 5EEQ SPR G7-B1 peptide 1.5 ± 0.01 μM[56]NA Replication proteinA 4NB3 FP Peptide-33 0.022 ± 0.005 μM[75]NA eIF4E 4BEA SPR sTIP-04 Stapled peptide 5 ± 0.7 [58]NA FP 11.5 ± 3.6 β-catenin 4DJS FP aStAx-35 13 ± 2.0 [76]NA hDcn-1 3TDZ ITC hCul1WHB: hDcn1P: Acetyl-hUbc121–12(5:9 Staple) 0.15 μM[77]NA Insulin 3KQ6 Receptor Binding Assays [HisA4, HisA8] insulin IGF-1R/0.05 ± 0.01 [54]NA IR/125 ± 18 ks-vFLIP 5LDE ITC spIKKƔ-Stapled peptide 30.4 ± 3.8 μM[52]NA TLE1 5MWJ ITC Peptide18 522 ± 39.6 [59]NA human IgG1 Fc 5U66 SPR LH1 ~1 ± 0.5 mM [57]NA

CaV β subunit 5V2P ITC AID-CAP Stapled peptide 5.1 ± 1.6 [78]NA 5V2Q ITC AID-CEN Stapled peptide 5.2 ± 1.5 [78]NA NCOA1 5Y7W – YL-2 – [79]NA Saccharomyces cerevisiae 5NXQ FP Sld5 CIP A2 0.32 ± 0.02 μM[80]NA 4HU6 – GCN4-p1(7b) – [81]NA CRPs (Plants) 5NGN – Lyba2 – [82]NA HIV-1 4NGH – SAH- MPER(671-683KKK)(q)pSer – [13]NA 4NHC – SAH-MPER(671-683KKK)(q) – [13]NA 4U6G – SAH-MPER(662-683KKK)(B,q) [13]NA 8HVP – Ua-I-OH 85548e – [83]NA 7HVP – JG-365 – [84]NA 2L6E Total buried surface NYAD-13 1 μMNA[85] 2JUK – GNB – NA [86] 1ZJ2 – HIV-1 frameshift site RNA – NA [87] 1PJY – HIV-1 frameshift inducing stem–loop RNA – NA [88] Brevibacillus Bacteria 4OZK – LS – [89]NA Zebraﬁsh MDM2/X 4N5T Biacore ATSP-7041 MDM2/0.91 [35]NA MDMX/2.31 Plasmodium falciparum 4MZJ – pGly[801–805] – [90]NA 4MZK – pGly[807-811] – [90]NA 4MZL – HSB myoA – [90]NA XRMV 4JGS – ɣ-XMRV TM retroviral fusion protein – [91]NA MPMV 4JF3 – β-MPMV TM retroviral fusion protein – [91]NA Salmonella 1Q5Z – SipA – [92]NA

(continued on next page) 272 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

Table 2 (continued)

Target PDB ID Binding Assay Peptide Kd (nM) Ref. X-ray NMR

Synthetic collagen 3P46 – SS1 – [93]NA EphA2-Sam/Ship2-Sam complex 6F7M MST S13ST Ship2-Sam/52.2 ± 0.7 μMNA[94] 6F7N MST S13ST (short) Ship2-Sam/No binding NA [94] 6F7O MST A5ST Ship2-Sam/No binding NA [94] Human Cul3-BTB 2MYL FP Cul349-68EN 620 ± 177 NA [95] 2MYM FP Cul349-68LA 305 ± 100 NA [95] SIV 2JTP – RNA stem-loop – NA [96] α-helical hairpin proteins 1EI0 – P8MTCP1 – NA [97] De novo proteins 2M7C – Cp-T2C3b – NA [98] 2M7D – (P12W)-T2C16b – NA [98]

regioisomer and four hydrophobic interactions were found with synthesize a highly potent stapled peptide (MCL-1 SAHBD), which selec- the protein surface residues: Leu54, Phe55, Gln59 and Met62. This tively binds MCL-1 and prevent it from suppressing the apoptosis path- mode of binding was similar to previously reported structure PDB: way and sensitizing caspase-dependent apoptosis within cancer cells. A 3V3B (described in Section 4.1) indicating that both staples are sited library of stapled alpha-helices of BCL-2 domain peptides was synthe- at the rim area of the p53-binding pocket, where Phe55 is the most im- sized based on the BH3 domain of human BCL-2 family and stapling portant residue (Fig. 13b). The proteolytic stability, cellular uptake and was located at the i,i + 4 positions using ring-closing metathesis toxicity of E1 peptide were evaluated, in which it showed high stability (RCM). To define the binding and specificity of BH3 helix and MCL-1 in a chymotrypsin assay, significant cellular permeability observed by alone, alanine scanning, site-direct mutagenesis and staple scanning confocal microscopy and did not show non-specific toxicity as deter- were performed; the results indicated that MCL-1 SAHBD has the mined in an LDH leakage assay. highest helicity ~90% and strongest binding, with a Kd of 10 nM as deter- mined by an FP assay. The complex structure of the stapled peptide with 4.3. Specific MCL-1 Stapled Peptide Inhibitor as Apoptosis Sensitizer in Can- MCL-1ΔNΔC was solved at 2.3 Å resolution (PDB 3MK8) and showed cer Cells that MCL-1 SAHBD is present in a helical conformation and interacts with the MCL-1 canonical BH3-binding pocket. The peptide α-helix The members of BCL-2 family known to have an anti-apoptotic role conserved residues L213, V216, G217, and V220 make a direct hydro- in cells are considered to be key pathogenic proteins in human diseases phobic contact with the MCL-1 interface that is consistent with many categorized by uncontrolled cell survival - such as cancer and autoim- BH3 domains. These hydrophobic interactions are reinforced by a salt mune disorders. The MCL-1 protein belongs to this family and supports bridge between MCL-1 SAHBD Asp218 and MCL-1ΔNΔCArg263 cell survival by trapping the apoptosis- inducing BCL-2 homology do- (Fig. 14). Interestingly, the hydrocarbon staple with alkene cis confor- main 3 (BH3) α-helix of pro-apoptotic BCL-2 family members. Cancer mation made a distinct hydrophobic contact with the edge of the cells utilize this physiological phenomenon by overexpressing anti- MCL-1 binding site. Moreover, the methyl group explores a groove com- apoptotic proteins to guarantee their immortality. As a result, develop- prising Gly262, Phe318, and Phe319 of MCL-1 and additional contact ing an inhibitor to block the hydrophobic pocket of the anti-apoptotic was found between the staple aliphatic side chain and the edge of the proteins from binding the BH3 α-helix could lead to the discovery of a main interaction site (Fig. 15). All of these structural evidence indicate successful drug. By mimicking BH3 α-helix, several small molecules the role of the staple in the high affinity binding of the peptide and its compounds were synthesized to inhibit anti-apoptotic proteins and ability to provide biological specificity toward MCL-1. This group also some are undergoing clinical trials (including ABT-263, obatoclax, and demonstrated the capacity of MCL-1 SAHBD to effectively sensitize mi- AT-101). Most target three or more anti-apoptotic member proteins, ex- tochondrial apoptosis in vitro using wild type and Bak−/− mitochon- cept the ABT-199 small molecule inhibitor, which has a high potency dria mouse models and in OPM2 cells by measuring the dissociation of and specificity to the BCL-2 protein with a Ki b 0.010 nM. ABT-199 native inhibitory MCL-1/BAK complexes using FP assay. In comparison was discovered through reverse engineering of navitoclax and keeping to ABT-199, the MCL-1 SAHBD stapled peptide shows good cell perme- similar hydrophobic interactions but modifying the electrostatic inter- ability and has the capacity to sensitize cancer cells to apoptosis when action with Arg103 (specific to BCL-2 not BCL-XL) [101]. Furthermore, tested on Jurkat T-cell leukemia and OPM2 cells, underscoring the clin- ABT-199 has antitumor activity against different cancers as non- ical relevance of these findings. However, the MCL-1 stapled peptide has Hodgkin's lymphoma (NHL) [101], refractory chronic lymphocytic not yet been evaluated in clinical trials. leukemia (CLL) [102,103], and BCL-2–dependent acute lymphoblastic leukemia (ALL) [101] in vitro. The same positive results were found 4.4. Stapled Peptides SP1, SP2 Inhibit Estrogen Receptor ERβ in vivo when ABT-199 was tested on a wide spectrum of xenograft mouse models harboring human hematological tumor (RS4;11), B cell Estrogen receptor (ER) is a steroid hormone receptor that belongs to lymphoma with the t(14,18) translocation [101] and mantel cell lym- the nuclear receptor (NR) superfamily class. In addition to the receptor's phoma (MCL) [101,104]. role in reproduction regulation, ER has a regulatory role in other path- Nonetheless, the topography of the binding groove and the amino ways in different systems such as the central nervous system, mainte- acids residues involved in the protein interaction of BH3 helix deter- nance of bone density and immunity. Thus, ER is an attractive target mine the specificity of the anti-apoptotic protein-binding partner. for diseases primarily in breast cancer, endometrial cancer and osteopo- Therefore, the need to discover an inhibitor that selectively targets the rosis [109–111]. Structurally the receptor existed in two isoforms (ERα interacting surface, which is large and complex, is essential. Walensky and ERβ), in which both have a similar domain organization - namely an and his group [68] selected MCL-1 as their research target, due to its N-terminal transactivation (AF1) domain, a well-conserved DNA bind- survival role in a wide-range of cancers and protein overexpression ing domain, and a C-terminal ligand-binding domain (LBD). Dependent that has been linked to the pathogenesis of diverse refractory cancers upon the bounded ligand, the ER receptor has two different states (including multiple myeloma, acute myeloid leukemia, melanoma and that induce changes in the structure, stability and interaction of the poor prognosis breast cancer [105–108]). This group was able to LED with a co-activator protein. When ER is in an agonist-bound A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 273

Table 3 The binding role of the staples in X-ray structures from RCSB-PDB.

Target PDB Peptide Cyclisation Method Kd (nM) Staple Interaction Ref. ID

Human MDM2 IYCR p53-WT Residues – 600 – [61] (15–29) 3V3B SAH-p53-8 Stapled RCM/i,i + 7 position 55 Hydrophobic contacts with Leu54, Phe55, Gly58, [62] peptide and Met62 of MDM2 4UMN M06 Stapled peptide RCM/i,i + 7 position 63 ± 17.8 Hydrophobic contacts with Leu54, Phe55 and more [63] closely with Gly 58 of MDM2 5AFG E1 Stapled peptide CuAAC cycloaddition 7.5 ± 0.7 Hydrophobic contacts with Leu54, Phe55, Gln59 [64] “Click-reaction” and Met62 of MDM2 4UE1 YS-1 Stapled peptide RCM/i,i + 4 position 9.9 ± 1.5 None [65] 4UD7 YS-2 Stapled peptide RCM/i,i + 4 position 7.4 ± 1.5 None [65] 5XXK M011 Stapled peptide RCM/i,i + 7 position 6.3 ± 2.9 Hydrophobic contacts with Leu54, Phe55 and more [66] closely with Gly 58 of MDM2 Zebraﬁsh MDM2/X 4N5T ATSP-7041 RCM/i,i + 7 position Mdm2/0.91 Van der Waals contacts with Lys47, Met50, His51, [35] MdmX/2.31 Gly54, Gln55, and Met58 of MDMX

MCL-1/BCL-2 3MK8 MCL-1 SAHBD Stapled RCM/i,i + 4 position 10 ± 3 Hydrophobic contacts with G262, F318, and F319 of [68] peptide MCL-1 5C3F BID-MM Stapled RCM/i,i + 4 position 153 ± 12 None [69] peptide 107 ± 29 5C3G BIM-MM Stapled RCM/i,i + 4 position 460 ± 232 None [69] peptide 5W89 SAH-MS1-18 Stapled RCM/i,i + 4 position 25 ± 7 None [70] peptide 5W8F SAH-MS1-14 Stapled RCM/i,i + 4 position 80 ± 5 None [70] peptide 5WHH D-NA-NOXA SAHB RCM/i,i + 7 position – None [71] Stapled peptide Estrogen Receptor 2YJD SP1 RCM/i,i + 4 position 1.99 μM Van der Waals contacts with Val307, Ile310, and [72] Leu490 of ERβ_LBD 2YJA SP2 RCM/i,i + 3 position 352 Van der Waals contacts with Val307, Ile310, and [72] Leu490 of ERβ_LBD 5DXB SRC2-SP1 RCM/i,i + 4 position 530 None [73] 5HYR SRC2-SP2 RCM/i,i + 4 position 42 None [73] 5DX3 SRC2-SP3 RCM/i,i + 4 position 39 None [73] 5DXE SRC2-SP4 RCM/i,i + 4 position – None [73] 5DXG SRC2-SP5 ––– [73] 5WGD SRC2-LP1 RCM/i,i + 4 position – Hydrophobic contacts between ILe689 and Leu693 [74] with the hydrophobic shelf of ERα 5WGQ SRC2-BCP1 Cross stitch (oleﬁn& – Sub-optimal hydrophobic interaction [74] lactam)/orthogonal position Aurora-A 5LXM Stapled TPX2 peptide RCM/i,i + 4 position 0.18 μM None [73] 10 Tankyrase 2 5BXO Cp4n2m3 Double-click Cycloaddition 0.6 ± 0.01 μM None [55] reaction/i,i + 4 position 5BXU Cp4n4m5 Double-click Cycloaddition 2.8 ± 0.1 μM None [55] reaction/i,i + 4 position Grb7 5D0J G7-B4NS peptide RCM+ Thioether/monocyclic 4.93 ± 0.03 μM None [56] peptide 5EEL G7-B4 peptide RCM+ Thioether/bicyclic peptide 0.83 ± 0.006 μM Close contacts with Met495, Asp496, Asp497 [56] backbone and sidechains of EF loop of Grb7-SH2 and Ile 518 of BG loop 5EEQ G7-B1 peptide RCM+ Thioether/bicyclic peptide 1.5 ± 0.01 μM Close contacts with Met495, Asp496, Asp497 [56] backbone and sidechains of EF loop of Grb7-SH2 and Ile 518 of BG loop Replication protein A 4NB3 Peptide-33 – 0.022 ± 0.005 – [75] μM eIF4E 4BEA sTIP-04 Stapled RCM/i,i + 4 position FP/11.5 ± 3.6 None [58] peptide SPR/5 ± 0.7 β-catenin 4DJS aStAx-35 RCM/i,i + 4 position 13 ± 2.0 None [76] hDcn-1 3TDZ hCul1WHB: hDcn1P: RCM/i,i + 4 position 0.15 μM None [77] Acetyl-hUbc121–12 (5:9 Staple) Insulin 3KQ6 [HisA4, HisA8] insulin – IGF-1R/0.05 ± – [54] 0.01 IR/125 ± 18 [52] ks-vFLIP 5LDE spIKKƔ-Stapled RCM/i,i + 4 position 30.4 ± 3.8 μM None [59] peptide TLE1 5MWJ Peptide18 RCM/i,i + 4 position 522 ± 39.6 None [57] Human 5U66 LH1 RCM/i,i + 7 position ~1 ± 0.5 mM None [78] IgG1 Fc

CaV β subunit 5V2P AID-CAP Stapled m-xylyl linker macrocyclization/i,i + 5.1 ± 1.6 None [78] peptide 5 position

CaV β subunit 5V2Q AID-CEN Stapled m-xylyl linker macrocyclization/i,i + 5.2 ± 1.5 None [75] peptide 4 position

(continued on next page) 274 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

Table 3 (continued)

Target PDB Peptide Cyclisation Method Kd (nM) Staple Interaction Ref. ID

Target PDB Peptide Cyclisation Method Kd (nM) Staple Interaction Ref. ID NCOA1 5Y7W YL-2 RCM/i,i + 4 position – None [79] Saccharomycs cerevisiae 5NXQ Sld5 CIP A2 Double-click Cycloaddition reaction 0.32 ± 0.02 μM None [80] (CuAAC)/i,i + 6 position 4HU6 GCN4-p1(7b) Oxime bridge (covalent – Internal polar contact between Gln4 and the U5 [81] cross-link)/i,i + 4 position carbonyl of the oxime bridge. HIV-1 4NGH SAH- MPER RCM/i,i + 4 position (R3-S5) – None [13]

(671-683KKK)(q)pSer

4NHC SAH-MPER(671-683KKK) RCM/i,i + 4 position (R3-S5) – None [13] (q)

4U6G SAH-MPER(662-683KKK) RCM/i,i + 4 position (R3-S5) None [13] (B,q) Plasmodium falciparum 4MZJ pGly[801–805] RCM/i,i + 4 position – Hydrophobic contact with Trp171 and Asp173 [90] 4MZK pGly[807-811] RCM/i,i + 4 position – Hydrophobic contact with Phe148, Leu168, Leu175 [90] an Ile202 Synthetic collagen 3P46 SS1 Double-click Cycloaddition reaction – None [93] (CuAAC) 2 + 1 strand click-reaction/C-terminal EphA2-Sam/Ship2-Sam 6F7M S13ST RCM/i,i + 4 position Ship2-Sam/52.2 None [94] complex ± 0.7 μM Human Cul3-BTB 2MYL Cul349-68EN RCM/i,i + 4 position 620 ± 177 None [95] 2MYM Cul349-68LA RCM/i,i + 4 position 305 ± 100 None [95] De novo proteins 2M7C Cp-T2C3b Gly-Gly linker – None [98] 2M7D (P12W)-T2C16b Gly-Gly linker – None [98]

conformation, a coactivator protein binding-groove is formed, con- clamp. Modeling of peptide inhibitors that bind to ER and work alloste- versely in the antagonist-bound conformation; the groove is lost. The rically could create a new class of NR-regulating drugs. Phillips and his co-activator binding-site is mediated by a short leucine-rich pentapep- group [72] designed and synthesized a series of these peptides, aiming tide, with the amino acid consensus sequence LXXXLL, known as the to bind and inhibit ER as pharmacological candidates. Two stapled NR box. This peptide was found to form amphipathic α-helices, in peptides known as SP1 and SP2 showed increased helicity as judged which the three conserved leucine residues are on the hydrophobic by CD. SP1 showed ~4 fold stronger binding to ER when compared face that binds to the coactivator-binding groove. On the other side of to unstapled peptides with a Kd of 1.99 μM, while SP2 peptide gave the binding site, the receptor surface has charged recognition residues 2-fold increase in binding relative to SP1 (Kd of 352 nM), as deter- that bind to the N and C-terminus of the helix, known as a charge mined by an SPR assay. The binding mode of both peptides followed

Leu54 Leu54

Phe55 Trp23

Leu26

Trp23 Phe19 Gly58

Met62

Fig. 11. Alignment of the SAH-p53-8 peptide (yellow, PDB 3V3B) and the native p53 peptide (cyan, PDB 1YCR). The MDM2 molecule is shown in surface representation. SAH-p53-8 peptide mimics the three pharmacophore residues (Phe19, Leu26, Trp23) in Fig. 12. A closer view of the SAH-p53-8 stapled peptide in a “closed” conformation the binding site in a similar manner to the native p53. The residues outside the Phe19- state. The MDM2 molecule is shown in surface representation, the peptide (yellow) Leu26 regions are not visible, indicating conformational flexibility in the bound state. and the staple (orange) in sticks. A hydrogen bond is formed between the indole Moreover, the whole helix of stapled peptide moves by ~1 Å and is rotated by 18°, nitrogen atom of the peptide helix and the carbonyl oxygen of Leu54 of MDM2 allowing the Trp23 indole ring to form a hydrogen bond with MDM2 Leu54 (green line). (green line). This H-bound is protected from solvent competition by the staple that Interestingly, Leu26 orientates itself in a distinct manner to that of the native p53 Leu26, lied directly over Met50-Lys64 helix (the rim of p53 binding site). In addition, the (moving by 2.7 Å toward the N-terminus of the peptide) and the side chain is flipped by staple intimately interacts with the protein surface and forms an extended approximately 180° to fill the same pocket space. This feature is not found in any other hydrophobic interface with Leu54, Phe55, Gly58, and Met62 of Mdm2. (For reported structure. (For interpretation of the references to color in this figure legend, interpretation of the references to colour in this figure legend, the reader is referred the reader is referred to the web version of this article.) to the web version of this article.) A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 275

Leu26 Phe19

N Trp23 N N N N N

Ac LTF N C EYWAQL N C S NH2 H H b) Gln59 O O Met62

Phe55

Leu54

Fig. 13. The E1-MDM2 complex high-resolution structure at 1.9 Å. a) top view of E1 stapled peptide (magenta, PDB 5AFG) aligned with the native peptide p53 (cyan, PDB 1YCR), revealing the typical mode of binding within the MDM2 hydrophobic pocket (grey surface) - placing the triad residues responsible for binding (P2he19, Trp23, Leu26) in the correct orientation to engage the MDM2 hotspots. The staple is found in anti regioisomer form and interacts with protein surface a similar mode as b) previously reported hydrocarbon SAH-p53-8 stapled peptide (PDB 3V3B), in that the stapled form four hydrophobic interactions with MDM2 surface residues (Leu54, Phe55, Gln59 and Met62, lime green), in which Phe55 is the most critical residue. The superimposition of the triazole-stapled E1 peptide with the correlated hydrocarbon-stapled p53 peptide (yellow, PDB 3V3B) suggests that both staples engage the same area that is located at the rim of the p53 binding pocket, on the Met50–Lys64 helix. The E1 stapled peptide is also shown in 2D for clarity (right). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) the same manner within the co-activator binding-site as shown by residue Asp538 rotates in toward the peptide, while it moves out in high-resolution structures at 1.9 Å for SP1 ERβ structure (PDB 1YJD), the co-activator peptide structure to adjust the Ile side chain into and 1.8 Å for the SP2 ERα structure (PDB 2YJA). The conserved NR the peptide-binding motif. Furthermore, Ile358 of the SP2 peptide box LXXLL recognition motif was replaced with the IXXS5L motif in adopts a distinct rotamer and packed closer than the second Leu of SP1; as expected the recognition site does not bind in the groove. Al- the coactivator peptide 2, which in turn allows the staple to span ternatively, the i,i + 4 hydrocarbon staple “at S5 position of the motif” over the C-terminal recognition motif with a 100° rotation toward was linked to the SP1 helix by RCM, which made comprehensive van the other side of the protein IL__LL contact site (Fig. 17b). der Waals contacts with the hydrophobic residues Val307, Ile310, and Leu490 of the co-activator protein binding groove (Fig. 16). As in the 5. Computational Approach for Staple Peptide Design SP1 ERβ structure, the same interactions for the staple with the hydrophobic groove of the coactivator site were found in the SP2 ERα Hydrocarbon stapled peptides are at a relatively early stage of devel- structure (Fig. 16). The reported structures demonstrate the impor- opment. Over the last decade important information about the effects of tance of designing stapled peptides that inhibit NRs and PPIs in staple position, staple structure, and peptide sequence on the activity of which stapling not only reinforces the helix conformational but also stapled peptides have become available [65]. Most of the reported sta- promotes staple interaction with the hydrophobic surface of the target pled peptides in the literature have classically been designed based ei- protein. Together this makes stapled peptides promising therapeutic ther on previous high-resolution structures or comprehensive alanine targets, even for the undruggable targets of traditional small- scanning studies [5,112]. On the other hand, staple positioning is usually molecule inhibitors. A comparison analysis between the stapled pep- optimized via chemical synthesis and biophysical characterization of tides SP1, SP2 and an earlier reported NR co-activator peptide 2 in every possible variant of a peptide construct. However, these methods complex with ERα (PDB 2QGT) revealed a quarter turn of the SP1 are by no means comprehensive and provide little insight about the be- helix that shifts the binding site residues by one position in the refer- havior of a stapled construct in living cells [113]. Moreover, experimen- ence recognition site (Fig. 17a). However, the SP2 stapled peptide tal characterization of the stapled peptides is neither economically showed conformational changes of Asp538 and Ile358 residues on viable nor feasible within a reasonable timeline, especially for long pep- the receptor site in contrast to the co-activator peptide 2. In SP2 tides, and is considered tedious and expensive [65,113]. 276 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

An example of a successful computational approach was the CCmut3 stapled peptide, a Bcr-Abl kinase agonist that effectively reduces the oncogenic potential of Bcr-Abl. By applying different in silico tools such as Chimera [114], AmberTools15 [115] and hydrogen mass repartitioning (HMR) for accelerating molecular dynamics (MD) simulations [113,116]; the authors were able to explore 64 peptide analogues with different possible positions of staple along peptide backbone. Such models can be used to characterize a wider range of possibilities than was possible experimentally, due to CCmut3 peptide length, which is considered long. Additionally, the length of the peptide introduces a high degree of conformational variability and opportunity to be targeted by intracellular proteolytic mechanisms. The authors concluded that computational methods can play a key role in the design process of therapeutics peptides, speciﬁcally in exploring an exceptionally large and diverse set of candidates in a short timeframe compared to experimental settings [113].

6. Conclusion

Inhibiting protein-protein interactions (PPI) has become a general strategy for interpreting the molecular logic associated with PPI networks, or for therapeutic applications. The later approach was explored Fig. 14. The crystal structure of MCL-1 SAHBD stapled peptide (slate helix) binding to the MCL-1ΔNΔC (light pink surface) interface at the canonical BH3-binding groove, solved at extensively over the last ten years by introducing a new class of targeted 2.3 Å resolution (PDB 3MK8). The peptide makes several hydrophobic interactions, inhibitors known as hydrocarbon-stapled peptides. Peptides originating including the hydrophobic residues Leu213, Val216, Gly217, and Val220 of MCL-1 from livestock and biological sources have low stability, short half-life Δ Δ SAHBD making direct contact with a hydrophobic cleft at the surface of MCL- 1 N C and unstable secondary-structures due to their sensitivity toward pro- (hot pink). The hydrophobic interaction are reinforced by a salt bridge between MCL-1 teolysis [1,16]. These impact peptide bioavailability and binding to SAHBD Asp218 and MCL-1ΔNΔC Arg263 (blue) and these residues also contribute to a hydrogen bond cluster that includes MCL-1ΔNΔC Asp256 and Asn260 (green). (For their intracellular target interfaces, making peptides a poor drug candi- interpretation of the references to colour in this figure legend, the reader is referred to date. However, small molecules are successful drug candidates due to the web version of this article.) their size, oral bioavailability and cell penetration. In addition, small molecules are more appropriate for small and compact protein inter- As a result, computational tools have become an essential part of the faces with a size range of 300–1000 Å [8] such as, BCL2 & BCL-xL pro- stapled peptide design process, because they help to rationalize practi- teins (Fig. 18a) [117], p53-MDM2 (Fig. 18b) [62,118] and Hsp90 cal/experimental observations, provide insight into molecular mecha- [119,120](Fig. 18c). Selected examples of these fruitful small- nisms of binding, and identify promising candidate peptides, thus molecules drugs are Obatoclax (GS-01570) which entered Phase II clin- reducing the need for extensive screening of peptide libraries. At the ical trials in patients with small-cell lung cancer [121–124]; Nutlin and same time, utilizing allows to efficiently build and characterize peptide its derivatives [125] led to the evolution of RG7112 as a first MDM2 in- candidates in silico. Thus, we are able to explore each possible staple lo- hibitor that entered clinical trials in advanced solid tumor patients in cation along the peptide backbone in order to ensure that each candi- 2007. This was followed by RO5503781 that entered Phase I clinical tri- date is considered in the search. All of these advantages reduce costs als in patients with advanced malignancies in 2011 and ended in 2014 and time that are necessary to design and optimize the lead stapled pep- (https://clinicaltrials.gov/ct2/show/record/NCT01462175). A last exam- tide for detailed experimental studies [65,113]. ple is a natural product (Geldanamycin (GM) (Fig. 18c)), which was the Several computational methods or techniques have been employed first molecule to inhibit Hsp90. Geldanamycin entered clinical trials and in stapled peptide design, including Energy minimization, Monte Carlo its clinical derivative, 17-AAG, reached phases I and II trials in patients (MC) simulation and Molecular dynamics (MD) [65]. with multiple myeloma, lymphoma, stage IV pancreatic cancer, non- small-cell lung cancer and solid tumors [126]. Despite the success of small-molecules to perturb different PPIs, these traditional inhibitors are not sufficient to cover large interfaces, which are more likely amenable to peptidomimetics. Gly262 Subsequently, synthetic and medicinal chemistry developments delivered stapling as a technique to overcome the limitation of native peptides in stability, resistance to proteolysis degradation, Phe319 specificity to targets and cell penetration. Depending on the size of the targeted interface, the affinity of the interaction and Phe318 the position of hot spots residues, different strategies have been generated to synthesize stapled peptides targeting major PPIs interfaces of previously undruggable protein networks. All- hydrocarbon stapled peptides lead to the discovery of new candidate drugs, combining the advantages of small-molecules and biologics. Examples of these interfaces are p53-MDM2/MDMX, BCL-2 family including MCL-1 BH3 domain, Estrogen receptor, Human

Fig. 15. The hydrocarbon staple of MCL-1 SAHBD peptide with an alkene functionality in immunodeficiency virus type 1 (HIV-1), kinases and Growth fac- the cis conformation (yellow stick) makes distinct hydrophobic contacts with the MCL- tor. As a result, stapling has found a unique therapeutic niche as 1ΔNΔC binding site border (light pink surface). A methyl group of the α,α-dimethyl an important class in the pharmaceutical field. Furthermore, sci- functionality engages with a groove consisting of MCL-1ΔNΔC Gly262, Phe318, and fi Phe319 residues (raspberry sticks). (For interpretation of the references to colour in this enti c technology innovation and novel chemistry methodologies figure legend, the reader is referred to the web version of this article.) broaden therapeutic peptide diversity and improve their A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 277

Leu490 Leu490

Val307 Val307

SP1 SP2 Ile310

Ile310

Fig. 16. The crystal structures of the SP1 ERβ (PDB 1YJD) and SP2 ERα (PDB 2YJA) complexes at 1.9 and 1.8 Å resolution, respectively. Van der Waal interactions (yellow dash lines) were found between both staples of SP1 and SP2 peptides and the hydrophobic residues on the surface of the co-activator binding site Val307, Ile310 and Leu490. ERβ and ERα proteins are shown in surface representation, while the staple of the both peptides and the interacting residues are in sticks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) pharmaceutical properties. In addition, advances in peptide affinities do not necessary translate to the target cell, as screening and computational biology will continue to support Wallbrecher and Okamoto demonstrated when applying stapling peptide drug discovery. It has been found that stapling increased to BimBH3 peptides to bind BCL-2 proteins and induce apoptosis peptide helicity and reinforces the α-helix confirmation as [131,132]. In agreement, PPI antagonists are usually evaluated shown in several studies, as well as enhancing specificity and using in vitro biochemical and biophysical assays like fluorescence binding affinity to the targeted protein interface [58,62,65,68,71]. polarization (FP), surface plasmon resonance (SPR) [25,29,133], However, some obstacles need to be overcome to improve thera- isothermal calorimetry (ITC) [32], enzyme-linked immunosorbent peutic properties, such as cellular penetration. In this case, new assay (ELIZA) [134]andfluorescence or bioluminescence reso- peptide drug delivery and formulation approaches are necessary nance excitation transfer (FRET/BRET) [135,136] to quantify their for this unique class of drugs, although some peptides show high binding potency and ability to disrupt the targeted PPI. Even permeability by endocytosis by optimizing helicity %, PI and hy- with the availability of fluorescent image-based assays such as drophobicity together [127]. Consequently, future studies should the proximity ligation in situ assay (P-LISA) [137,138]andtwo/ focus on the factors that promote cellular uptake and endosomal three hybrid (F2H and F3H) assays [99,139] to assess PPI antago- release in stapled peptide design. Another challenge that must nists in real-time within the cells; none can reveal whether be overcome is the oral availability of peptide therapeutics, these antagonists can cross the cell membrane effectively in a na- which could be boosted by increasing drug stability in the gastro- tive intracellular environment. To overcome this issue, the ReBiL intestinal tract, again by formulating peptide penetration with en- platform has been developed by Li and co-workers to detect hancers or congregating with carrier molecules or nanoparticles weak PPI interactions and mechanisms of drug action in living [128–130]. Finally, in some cases, stapled peptides with high cells. It has been successfully applied on antagonists of p53:

a) b)

Asp538

Ile358

Fig. 17. Comparison analysis of a) SP1 (lime green helix) and b) SP2 (aquamarine helix) stapled peptides in relation to previously reported NR co-activator peptide 2 (light magenta helix, PDB 2QGT). a) SP1 stapled peptide exhibited a quarter turn with respect to the co-activator peptide locating the hydrophobic staple to the recognition site position. While b) SP2 rotates differently and packs tighter than the coactivator peptide 2 does. Additionally, two residues in the receptor site (Asp538 and Ile358) induce conformational changes bridging the staple of SP2 helix to the C-terminus site of the recognition motif that is rotated by 100° toward the other side of the protein IL__LL contact site. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) 278 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

b) c)

Fig. 18. Crystal structures of successful small molecules inhibiting drug-target PPIs that have entered clinical trials. a) ABT-737 (pink sticks) binds to BCL-xL (PDB 2YXJ) with nanomolar binding affinity, b) Nutlin-2 (green sticks), is one of the first identified potent MDM2–p53 inhibitors and is shown bound to the N-terminal domain of MDM2 (PDB 1RV1) and c) geldanamycin (GM) (light blue stick) in complex with Hsp90 (PDB 1YET), considered the first Hsp90 inhibitor to enter clinical trials. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

MDM2 PPI, including small molecule Nutlin-3a and three stapled binds and which interactions are involved. Importantly, two ques- peptides (SAHBp53-8, ATSB-7104 and sMTide-02). They revealed tions need to be tackled in current research: Firstly, can a stapled that potent binding in vitro does not necessarily correlate with peptide with signiﬁcant in vitro results penetrate the cell membrane higher intracellular PPI disruption activity. This emphasizes the toward the target PPI complex? Secondly, if it reaches the target PPI, importance of using an assay like ReBiL to analyze directly the dis- will this peptide exert the expected bioactivity in vivo? This question ruption of target PPI within cells [140]. could be addressed with future research aiming to tailored bioactive Interestingly, relatively few reported high-resolution structures of therapeutic peptides with high permeability and increased precision stapled peptide in complex with their target interface reveal a direct in- to PPIs within the targeted cell using advanced medicinal and syn- teraction between the staple itself and residues of the protein interfaces thetic chemistry in parallel with computational and drug delivery

(Table 2). A phenomenon is observed with the MCL-1 SAHBD/MCL-1, approaches. SAH-p53-8/MDM2, E1/MDM2, SP1/ERβ and SP2/ERα complexes, in which staples make additional hydrophobic interaction with protein surface residues that fix the peptide in a bound state within the target Acknowledgment pocket. Additionally, the staple can play a role in peptide penetration and bioactivity [127]. This research has been supported to (AD) by the National Institute of Taken together, hydrocarbon-stapled peptides are a fertile ground Health (NIH) (2R01GM097082-05), the European Lead Factory (IMI) for drug discovery. However, the development of highly cell- under grant agreement number 115489, the Qatar National Research permeable and bioactive peptides is a challenging task that includes Foundation (NPRP6-065-3-012). Moreover funding was received several phases. Starting from the correct design of the peptide, through ITN “Accelerated Early stage drug dIScovery” (AEGIS, grant screening of different stapling positions and/or point mutations, mea- agreement No 675555) and, COFUND ALERT (grant agreement No suring binding affinities and testing specificity toward the target pro- 665250) and KWF Kankerbestrijding grant (grant agreement No tein interface, structural and computational analysis should be 10504). A.A was supported by Qatar leadership program, Qatar performed in order to understand how the peptide and/or staple foundation. A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 279

References therapeutic. Proc Natl Acad Sci 2010;107:14093–8. https://doi.org/10.1073/pnas. 1002713107. [1] Craik DJ, Fairlie DP, Liras S, Price D. The future of peptide-based drugs. Chem Biol [31] Bird GH, Christian Crannell W, Walensky LD. Chemical synthesis of hydrocarbon- Drug Des 2013;81:136–47. https://doi.org/10.1111/cbdd.12055. stapled peptides for protein interaction research and therapeutic targeting. Curr – [2] Rosenblum D, Peer D. Omics-based nanomedicine: the future of personalized on- Protoc Chem Biol 2011;3:99 117. https://doi.org/10.1002/9780470559277. cology. Cancer Lett 2014;352:126–36. https://doi.org/10.1016/j.canlet.2013.07.029. ch110042. [3] Ruffner H, Bauer A, Bouwmeester T. Human protein–protein interaction networks [32] Walensky LD, Bird GH. Hydrocarbon-stapled peptides: principles, practice, and – and the value for drug discovery. Drug Discov Today 2007;12:709–16. https://doi. progress. J Med Chem 2014;57:6275 88. https://doi.org/10.1021/jm4011675. – org/10.1016/J.DRUDIS.2007.07.011. [33] Wang Y, Chou DH. A thiol Ene coupling approach to native peptide stapling and – [4] Yan C, Wu F, Jernigan RL, Dobbs D, Honavar V. Characterization of protein- macrocyclization ** Angewandte. Angew Chem Int Ed 2015;54:10931 4. https:// protein interfaces. Protein J 2008;27:59–70. https://doi.org/10.1007/s10930-007- doi.org/10.1002/anie.201503975. 9108-x. [34] Kneissl S, Loveridge EJ, Williams C, Crump MP, Allemann RK. Photocontrollable [5] Verdine GL, Hilinski GJ. Stapled peptides for intracellular drug targets. . 1st ed., vol. peptide-based switches target the anti-apoptotic protein Bcl-x L. Chem Bio Chem – 503Elsevier Inc; 2012. https://doi.org/10.1016/B978-0-12-396962-0.00001-X. 2008;9:3046 54. https://doi.org/10.1002/cbic.200800502. α− [6] Pérot S, Sperandio O, Miteva MA, Camproux AC, Villoutreix BO. Druggable pockets [35] Chang YS, Graves B, Guerlavais V, Tovar C, Packman K, To K-H, et al. Stapled he- and binding site centric chemical space: a paradigm shift in drug discovery. Drug lical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for – Discov Today 2010;15:656–67. https://doi.org/10.1016/j.drudis.2010.05.015. p53-dependent cancer therapy. Proc Natl Acad Sci 2013;110:E3445 54. https:// [7] Ivanov AA, Khuri FR, Fu H. Targeting protein-protein interactions as an anticancer doi.org/10.1073/pnas.1303002110. strategy. Trends Pharmacol Sci 2013;34:393–400. https://doi.org/10.1016/j.tips. [36] Hilinski GJ, Kim Y-W, Hong J, Kutchukian PS, Crenshaw CM, Berkovitch SS, et al. α 2013.04.007. Stitched -helical peptides via bis ring-closing metathesis; 2014. https://doi.org/ [8] Smith MC, Gestwicki JE. Features of protein–protein interactions that translate into 10.1021/ja505141j. potent inhibitors: topology, surface area and affinity. Expert Rev Mol Med 2012;14: [37] Felix AM, Heimer EP, Wang CT, Lambros TJ, Fournier A, Mowles TF, et al. Synthesis, e16. https://doi.org/10.1017/erm.2012.10. biological activity and conformational analysis of cyclic GRF analogs. Int J Pept Pro- – [9] Fang Z, Song Y, Zhan P, Zhang Q, Liu X. Conformational restriction: an effective tac- tein Res 1988;32:441 54. https://doi.org/10.1111/j.1399-3011.1988.tb01375.x. fi tic in ‘follow-on’-based drug discovery. Future Med Chem 2014;6:885–901. https:// [38] Green eld N, Fasman GD. Computed circular dichroism spectra for the evaluation – doi.org/10.4155/fmc.14.50. of protein conformation. Biochemistry 1969;8:4180 4116. doi:https://doi.org/10. [10] Gilson MK, Zhou H-X. Calculation of protein-ligand binding affinities. Annu Rev 1021/bi00838a031. Biophys Biomol Struct 2007;36:21–42. https://doi.org/10.1146/annurev.biophys. [39] Shepherd NE, Hoang HN, Abbenante G, Fairlie DP. Single turn peptide alpha helices – 36.040306.132550. with exceptional stability in water. J Am Chem Soc 2005;127:2974 83. https://doi. [11] Bakail M, Ochsenbein F. Targeting protein-protein interactions, a wide open field org/10.1021/ja0456003. for drug design. C R Chim 2016;19:19–27. https://doi.org/10.1016/j.crci.2015.12. [40] Khoo KK, Wilson MJ, Smith BJ, Zhang M-M, Gulyas J, Yoshikami D, et al. Lactam- μ 004. stabilized helical analogues of the analgesic -conotoxin KIIIA. J Med Chem 2011; [12] Okamoto T, Segal D, Zobel K, Fedorova A, Yang H, Fairbrother WJ, et al. Further in- 54:7558. https://doi.org/10.1021/JM200839A. sights into the effects of pre-organizing the BimBH3 Helix. ACS Chem Biol 2014;9: [41] Moses JE, Moorhouse AD, Kolb HC, Finn MG, Sharpless KB, Kolb HC, et al. The grow- – 838–9. https://doi.org/10.1021/cb400638p. ing applications of click chemistry. Chem Soc Rev 2007;36:1249 62. https://doi. [13] Bird GH, Irimia A, Ofek G, Kwong PD, Wilson IA, Walensky LD. Stapled HIV-1 pep- org/10.1039/B613014N. tides recapitulate antigenic structures and engage broadly neutralizing antibodies. [42] Scrima M, Le Chevalier-Isaad A, Rovero P, Papini AM, Chorev M, D'Ursi AM. Cui cat- Nat Struct Mol Biol 2014;21:1058–67. https://doi.org/10.1038/nsmb.2922. alyzed azide-alkyne intramolecular i-to-(i+4) side-chain-to-SideChain cyclization [14] Lau YH, De Andrade P, Wu Y, Spring DR. Peptide stapling techniques based on dif- promotes the formation of Helix-like secondary structures. European J Org Chem – ferent macrocyclisation chemistries. Chem Soc Rev 2015;44:91–102. https://doi. 2010;2010:446 57. https://doi.org/10.1002/ejoc.200901157. org/10.1039/c4cs00246f. [43] Kawamoto SA, Coleska A, Ran X, Yi H, Yang CY, Wang S. Design of triazole-stapled α β [15] Jenssen H, Aspmo SI. Serum stability of peptides. Methods Mol Biol 2008;494: BCL9 -helical peptides to target the -catenin/B-cell CLL/lymphoma 9 (BCL9) – 177–86. https://doi.org/10.1007/978-1-59745-419-3_10. protein-protein interaction. J Med Chem 2012;55:1137 46. https://doi.org/10. [16] Tsomaia N. Peptide therapeutics: targeting the undruggable space. Eur J Med Chem 1021/jm201125d. 2015;94:459–70. https://doi.org/10.1016/j.ejmech.2015.01.014. [44] Madden MM, Muppidi A, Li Z, Li X, Chen J, Lin Q. Synthesis of cell-permeable sta- [17] Jubb H, Higueruelo AP, Winter A, Blundell TL. Structural biology and drug discovery pled peptide dual inhibitors of the p53-Mdm2/Mdmx interactions via photoin- – for protein-protein interactions. Trends Pharmacol Sci 2012;33:241–8. https://doi. duced cycloaddition. Bioorg Med Chem Lett 2011;21:1472 5. https://doi.org/10. org/10.1016/j.tips.2012.03.006. 1016/j.bmcl.2011.01.004. [18] Lau JL, Dunn MK. Therapeutic peptides: historical perspectives, current develop- [45] Jackson DY, King DS, Chmielewski J, Singh S, Schultz PG. General approach to the – ment trends, and future directions. Bioorg Med Chem 2018;26:2700–7. https:// synthesis of short .alpha.-helical peptides. J Am Chem Soc 1991;113:9391 2. doi.org/10.1016/j.bmc.2017.06.052. https://doi.org/10.1021/ja00024a067. α [19] Schafmeister CE, Po J, Verdine GL. An all-hydrocarbon cross-linking system for en- [46] Haney CM, Loch MT, Horne WS. Promoting peptide -helix formation with dy- hancing the helicity and metabolic stability of peptides [8]. J Am Chem Soc 2000; namic covalent oxime side-chain cross-links. Chem Commun 2011;47:10915. 122:5891–2. https://doi.org/10.1021/ja000563a. https://doi.org/10.1039/c1cc12010g. [20] Tyndall JDA, Nall T, Fairlie DP. Proteases universally recognize beta strands in their [47] Phelan JC, Skelton NJ, Braisted AC, McDowell RS. A general method for constraining α – active sites, vol. 105; 2005; 973–1000. https://doi.org/10.1021/cr040669e. short peptides to an -helical conformation. J Am Chem Soc 1997;119:455 60. [21] Nowick JS. Exploring β-sheet structure and interactions with chemical model sys- https://doi.org/10.1021/ja9611654. fl tems. Acc Chem Res 2008;41:1319–30. https://doi.org/10.1021/ar800064f. [48] Spokoyny AM, Zou Y, Ling JJ, Yu H, Lin Y-S, Pentelute BL. A Per uoroaryl-cysteine S [22] Harrison RS, Shepherd NE, Hoang HN, Ruiz-Gomez G, Hill TA, Driver RW, et al. N Ar chemistry approach to unprotected peptide stapling. J Am Chem Soc 2013; – Downsizing human, bacterial, and viral proteins to short water-stable alpha helices 135:5946 9. https://doi.org/10.1021/ja400119t. that maintain biological potency. Proc Natl Acad Sci 2010;107:11686–91. https:// [49] Brunel FM, Dawson PE. Synthesis of constrained helical peptides by thioether liga- – doi.org/10.1073/pnas.1002498107. tion: application to analogs of gp41. Chem Commun 2005:2552 4. https://doi.org/ [23] Kim Y-W, Grossmann TN, Verdine GL. Synthesis of all-hydrocarbon stapled α- 10.1039/b419015g. helical peptides by ring-closing olefin metathesis. Nat Protoc 2011;6:761–71. [50] Brunel FM, Zwick MB, Cardoso RMF, Nelson JD, Wilson IA, Burton DR, et al. https://doi.org/10.1038/nprot.2011.324. Structure-function analysis of the epitope for 4E10, a broadly neutralizing human fi [24] Blackwell HE, Grubbs RH. Highly efficient synthesis of covalently cross-linked pep- immunode ciency virus type 1 antibody. J Virol 2006;80:1680. https://doi.org/ tide helices by ring-closing metathesis. Angew Chem Int Ed 1998;37:3281–4. 10.1128/JVI.80.4.1680-1687.2006. https://doi.org/10.1002/(SICI)1521-3773(19981217)37:23b3281::AID- [51] RCSB. Protein data-bank. n.d www.rcsb.org. γ ANIE3281N3.0.CO;2-V. [52] Briggs LC, Chan AWE, Davis CA, Whitelock N, Hotiana HA, Baratchian M, et al. IKK - [25] Walensky LD, Kung AL, Escher I, Malia TJ, Barbuto S, Wright RD, et al. Activation of mimetic peptides block the resistance to apoptosis associated with Kaposi's sar- apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 2004;305. https:// coma-associated herpesvirus infection. J Virol 2017;91. https://doi.org/10.1128/ – doi.org/10.1126/SCIENCE.1099191 80-. 1466–70. JVI.01170-17 e01170 17. [26] Ahrens VM, Bellmann-Sickert K, Beck-Sickinger AG. Peptides and peptide conju- [53] Rennie YK, McIntyre PJ, Akindele T, Bayliss R, Jamieson AG. A TPX2 Proteomimetic fi gates: therapeutics on the upward path. Future Med Chem 2012;4:1567–86. has enhanced af nity for Aurora-a due to hydrocarbon stapling of a Helix. ACS – https://doi.org/10.4155/fmc.12.76. Chem Biol 2016;11:3383 90. https://doi.org/10.1021/acschembio.6b00727. [27] Miller MJ, Foy KC, Kaumaya PTP. Cancer immunotherapy: present status, future [54] Phillips NB, Wan ZL, Whittaker L, Hu SQ, Huang K, Hua QX, et al. Supramolecular perspective, and a new paradigm of peptide immunotherapeutics. Discov Med protein engineering: design of zinc-stapled insulin hexamers as a long – 2013;15(82):166–76. acting depot. J Biol Chem 2010;285:11755 9. https://doi.org/10.1074/jbc.C110. [28] Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M. Synthetic therapeutic peptides: 105825. science and market. Drug Discov Today 2010;15:40–56. https://doi.org/10.1016/J. [55] Xu W, Lau YH, Fischer G, Tan YS, Chattopadhyay A, De La Roche M, et al. DRUDIS.2009.10.009. Macrocyclized extended peptides: inhibiting the substrate-recognition domain of – [29] Bernal F, Tyler AF, Korsmeyer SJ, Walensky LD, Verdine GL. Reactivation of the p53 Tankyrase. J Am Chem Soc 2017;139:2245 56. https://doi.org/10.1021/jacs. tumor suppressor pathway by a stapled p53 peptide. J Am Chem Soc 2007;129: 6b10234. 2456–7. https://doi.org/10.1021/ja0693587. [56] Gunzburg MJ, Kulkarni K, Watson GM, Ambaye ND, Del Borgo MP, Brandt R, et al. [30] Bird GH, Madani N, Perry AF, Princiotto AM, Supko JG, He X, et al. Hydrocarbon Unexpected involvement of staple leads to redesign of selective bicyclic peptide in- double-stapling remedies the proteolytic instability of a lengthy peptide hibitor of Grb7. Sci Rep 2016;6:27060. https://doi.org/10.1038/srep27060. 280 A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281

[57] Ultsch M, Braisted A, Maun HR, Eigenbrot C. 3-2-1: structural insights from step- immunodeficiency virus type 1 protease complexed with a hydroxyethylene- wise shrinkage of a three-helix fc-binding domain to a single helix. Protein Eng based inhibitor. Biochemistry 1991;30:1600–9. https://doi.org/10.1021/ Des Sel 2017;30:619–25. https://doi.org/10.1093/protein/gzx029. bi00220a023. [58] Lama D, Quah ST, Verma CS, Lakshminarayanan R, Beuerman RW, Lane DP, et al. [84] Swain AL, Miller MM, Green J, Rich DH, Schneider J, Kent SB, et al. X-ray crystallo- Rational optimization of conformational effects induced by hydrocarbon staples graphic structure of a complex between a synthetic protease of human immunode- in peptides and their binding interfaces. Sci Rep 2013;3:3451. https://doi.org/10. ficiency virus 1 and a substrate-based hydroxyethylamine inhibitor. Proc Natl Acad 1038/srep03451. Sci U S A 1990;87 (8805–9. doi:2247451). [59] McGrath S, Tortorici M, Drouin L, Solanki S, Vidler L, Westwood I, et al. Structure- [85] Bhattacharya S, Zhang H, Debnath AK, Cowburn D. Solution structure of a hydrocar- enabled discovery of a stapled peptide inhibitor to target the oncogenic transcrip- bon stapled peptide inhibitor in complex with monomeric C-terminal domain of tional repressor TLE1. Chem A Eur J 2017;23:9577–84. https://doi.org/10.1002/ HIV-1 capsid. J Biol Chem 2008;283:16274–8. https://doi.org/10.1074/jbc. chem.201700747. C800048200. [60] Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, et al. A human [86] Staple DW, Venditti V, Niccolai N, Elson-Schwab L, Tor Y, Butcher SE. protein-protein interaction network: a resource for annotating the proteome. Cell Guanidinoneomycin B recognition of an HIV-1 RNA helix. ChemBioChem 2008;9: 2005;122:957–68. https://doi.org/10.1016/j.cell.2005.08.029. 93–102. https://doi.org/10.1002/cbic.200700251. [61] Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, et al. Structure of [87] Staple DW, Butcher SE. Solution structure and thermodynamic investigation of the the MSM2 oncoprotein bound to the p53 tumor suppressor transactivation do- HIV-1 frameshift inducing element. J Mol Biol 2005;349:1011–23. https://doi.org/ main. Science 1996;274 80-. (948–53). 10.1016/j.jmb.2005.03.038. [62] Baek S, Kutchukian PS, Verdine GL, Huber R, Holak TA, Lee KW, et al. Structure of [88] Staple DW, Butcher SE. Solution structure of the HIV-1 frameshift inducing stem- the stapled p53 peptide bound to Mdm2. J Am Chem Soc 2012;134:103–6. loop RNA. Nucleic Acids Res 2003;31:4326–31. https://doi.org/10.1093/nar/ https://doi.org/10.1021/ja2090367. gkg654. [63] Chee SMQ, Wongsantichon J, Soo Tng Q, Robinson R, Joseph TL, Verma C, et al. [89] Singh PK, Solanki V, Sharma S, Thakur KG, Krishnan B, Korpole S. The intramolecu- Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2. PLoS lar disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a One 2014;9:e104914. https://doi.org/10.1371/journal.pone.0104914. human defensin-like structural module. FEBS J 2015;282:203–14. https://doi.org/ [64] Lau YH, Wu Y, Rossmann M, Tan BX, De Andrade P, Tan YS, et al. Double strain-pro- 10.1111/febs.13129. moted macrocyclization for the rapid selection of cell-active stapled peptides. [90] Douse CH, Maas SJ, Thomas JC, Garnett JA, Sun Y, Cota E, et al. Crystal structures of Angew Chem Int Ed 2015;54:15410–3. https://doi.org/10.1002/anie.201508416. stapled and hydrogen bond surrogate peptides targeting a fully buried protein- [65] Tan YS, Reeks J, Brown CJ, Thean D, Ferrer Gago FJ, Yuen TY, et al. Benzene probes in helix interaction. ACS Chem Biol 2014;9:2204–9. https://doi.org/10.1021/ molecular dynamics simulations reveal novel binding sites for ligand design. J Phys cb500271c. Chem Lett 2016;7:3452–7. https://doi.org/10.1021/acs.jpclett.6b01525. [91] Aydin H, Cook JD, Lee JE. Crystal structures of Beta- and Gammaretrovirus fusion [66] Chee SMQ, Wongsantichon J, Siau J, Thean D, Ferrer F, Robinson RC, et al. Structure- proteins reveal a role for electrostatic stapling in viral entry. J Virol 2014;88: activity studies of Mdm2/Mdm4- binding stapled peptides comprising non- natural 143–53. https://doi.org/10.1128/JVI.02023-13. amino acids. PLoS One 2017;12:e0189379. https://doi.org/10.1371/journal.pone. [92] Lilic M, Galkin VE, Orlova A, VanLoock MS, Egelman EH, Stebbins CE. Actin by sta- 0189379. pling filaments with nonglobular protein arms. Science 2003;301 80-. (1918–21). [67] Li X, Lu W. No title. To Be Published 2018. [93] Byrne C, McEwan PA, Emsley J, Fischer PM, Chan WC. End-stapled homo and hetero [68] Stewart ML, Fire E, Keating AE, Walensky LD. The MCL-1 BH3 helix is an exclusive collagen triple helices: a click chemistry approach. Chem Commun 2011;47: MCL-1 inhibitor and apoptosis sensitizer. Nat Chem Biol 2010;6:595–601. https:// 2589–91. https://doi.org/10.1039/c0cc04795c. doi.org/10.1038/nchembio.391. [94] Mercurio FA, Pirone L, Di Natale C, Marasco D, Pedone EM, Leone M. Sam domain- [69] Miles JA, Yeo DJ, Rowell P, Rodriguez-Marin S, Pask CM, Warriner SL, et al. Hydro- based stapled peptides: structural analysis and interaction studies with the Sam carbon constrained peptides-understanding preorganisation and binding affinity. domains from the EphA2 receptor and the lipid phosphatase Ship2. Bioorg Chem Chem Sci 2016;7:3694–702. https://doi.org/10.1039/c5sc04048e. 2018;80:602–10. https://doi.org/10.1016/j.bioorg.2018.07.013. [70] Rezaei Araghi R, Bird GH, Ryan JA, Jenson JM, Godes M, Pritz JR, et al. Iterative op- [95] De Paola I, Pirone L, Palmieri M, Balasco N, Esposito L, Russo L, et al. Cullin3 - BTB timization yields mcl-1–targeting stapled peptides with selective cytotoxicity to interface:a novel target for stapled peptides. PLoS One 2015;10:1–21. https://doi. mcl-1–dependent cancer cells. Proc Natl Acad Sci 2018;201712952. https://doi. org/10.1371/journal.pone.0121149. org/10.1073/pnas.1712952115. [96] Marcheschi RJ, Staple DW, Butcher SE. Programmed ribosomal frameshifting in SIV [71] Harvey EP, Seo HS, Guerra RM, Bird GH, Dhe-Paganon S, Walensky LD. Crystal is induced by a highly structured RNA stem-loop. J Mol Biol 2007;373:652–63. structures of anti-apoptotic BFL-1 and Its complex with a covalent stapled peptide https://doi.org/10.1016/j.jmb.2007.08.033. inhibitor. Structure 2018;26:153–60 e4 https://doi.org/10.1016/j.str.2017.11.016. [97] Barthe P, Roumestand C, Rochette S, Vita C. Synthesis and NMR solution structure [72] Phillips C, Roberts LR, Schade M, Bazin R, Bent A, Davies NL, et al. Design and struc- of an α-helical hairpin stapled with two disulfide bridges. Protein Sci 2000;9: ture of stapled peptides binding to estrogen receptors. J Am Chem Soc 2011;133: 942–55. https://doi.org/10.1110/ps.9.5.942. 9696–9. https://doi.org/10.1021/ja202946k. [98] Byrne A, Kier BL, Williams DV, Scian M, Andersen NH. Circular permutation of the [73] Speltz TE, Fanning SW, Mayne CG, Fowler C, Tajkhorshid E, Greene GL, et al. Stapled Trp-cage: fold rescue upon addition of a hydrophobic staple. RSC Adv 2013;3: peptides with γ-methylated hydrocarbon chains for the estrogen receptor/coacti- 19824–9. https://doi.org/10.1039/c3ra43674h. vator interaction. Angew Chem Int Ed Engl 2016;55:4252–5. https://doi.org/10. [99] Brown CJ, Quah ST, Jong J, Goh AM, Chiam PC, Khoo KH, et al. Stapled peptides with 1002/anie.201510557. improved potency and specificity that activate p53. ACS Chem Biol 2013;8:506–12. [74] Speltz TE, Mayne CG, Fanning SW, Siddiqui Z, Tajkhorshid E, Greene GL, et al. A https://doi.org/10.1021/cb3005148. “cross-stitched” peptide with improved helicity and proteolytic stability. Org [100] Thean D, Ebo JS, Luxton T, Lee XC, Yuen TY, Ferrer FJ, et al. Enhancing specificdis- Biomol Chem 2018;16:3702–6. https://doi.org/10.1039/c8ob00790j. ruption of intracellular protein complexes by hydrocarbon stapled peptides using [75] Frank AO, Vangamudi B, Feldkamp MD, Souza-Fagundes EM, Luzwick JW, Cortez D, lipid based delivery. Sci Rep 2017;7:1–11. https://doi.org/10.1038/s41598-017- et al. Discovery of a potent stapled helix peptide that binds to the 70N domain of 01712-5. replication protein a. J Med Chem 2014;57:2455–61. https://doi.org/10.1021/ [101] Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al. ABT-199, a jm401730y. potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing [76] Grossmann TN, Yeh JT-H, Bowman BR, Chu Q, Moellering RE, Verdine GL. Inhibition platelets. Nat Med 2013;19:202–8. https://doi.org/10.1038/nm.3048. of oncogenic Wnt signaling through direct targeting of -catenin. Proc Natl Acad Sci [102] Singh RR, Kim JE, Davuluri Y, Drakos E, Cho-Vega JH, Amin HM, et al. Hedgehog sig- 2012;109:17942–7. https://doi.org/10.1073/pnas.1208396109. naling pathway is activated in diffuse large B-cell lymphoma and contributes to [77] Scott DC, Monda JK, Bennett EJ, Harper JW, Schulman BA. N-terminal acetylation tumor cell survival and proliferation. Leukemia 2010;24:1025–36. https://doi.org/ acts as an avidity enhancer within an interconnected multiprotein complex. Sci- 10.1038/leu.2010.35. ence 2011;334. https://doi.org/10.1126/science.1209307 80-. 674–8. [103] Vogler M, Dinsdale D, Dyer MJS, Cohen GM. ABT-199 selectively inhibits BCL2 but [78] Findeisen F, Campiglio M, Jo H, Abderemane-Ali F, Rumpf CH, Pope L, et al. Stapled not BCL2L1 and efficiently induces apoptosis of chronic lymphocytic leukaemic voltage-gated Calcium Channel (CaV) α-interaction domain (AID) peptides act as cells but not platelets. Br J Haematol 2013;163:139–42. https://doi.org/10.1111/ selective protein-protein interaction inhibitors of CaVFunction. ACS Chem Nerosci bjh.12457. 2017;8:1313–26. https://doi.org/10.1021/acschemneuro.6b00454. [104] Vandenberg CJ, Cory S. ABT-199, a new Bcl-2–specificBH3mimetic,hasinvivoef- [79] Lee Y, Yoon H, Hwang SM, Shin MK, Lee JH, Oh M, et al. Targeted inhibition of the ficacy against aggressive Myc-driven mouse lymphomas without provoking NCOA1/STAT6 protein-protein interaction. J Am Chem Soc 2017;139:16056–9. thrombocytopenia. Blood 2013;121:2285–8. https://doi.org/10.1182/blood-2013- https://doi.org/10.1021/jacs.7b08972. 01-475855. [80] Wu Y, Villa F, Maman J, Lau YH, Dobnikar L, Simon AC, et al. Targeting the genome- [105] Zhang B, Gojo I, Fenton RG. Myeloid cell factor-1 is a critical survival factor for mul- stability hub Ctf4 by stapled-peptide design. Angew Chem Int Ed 2017;56: tiple myeloma. Blood 2002;99:1885–93. https://doi.org/10.1182/blood.V99.6.1885. 12866–72. https://doi.org/10.1002/anie.201705611. [106] Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, et al. Mecha- [81] Haney CM, Horne WS. Oxime side-chain cross-links in an α-helical coiled-coil pro- nisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in tein: structure, thermodynamics, and folding-templated synthesis of bicyclic spe- acute myeloid leukemia. Cancer Cell 2006;10:375–88. https://doi.org/10.1016/j. cies. Chem A Eur J 2013;19:11342–51. https://doi.org/10.1002/chem.201300506. ccr.2006.10.006. [82] Tan WL, Wong KH, Lei J, Sakai N, Tan HW, Hilgenfeld R, et al. Lybatides from Lycium [107] Boisvert-Adamo K, Longmate W, Abel EV, Aplin AE. Mcl-1 is required for melanoma barbarum contain an unusual cystine-stapled helical peptide scaffold. Sci Rep cell resistance to Anoikis. Mol Cancer Res 2009;7:549–56. https://doi.org/10.1158/ 2017;7:1–11. https://doi.org/10.1038/s41598-017-05037-1. 1541-7786.MCR-08-0358. [83] Jaskólski M, Wlodawer A, Tomasselli AG, Sawyer TK, Staples DG, Heinrikson RL, [108] Ding Q, He X, Xia W, Hsu JM, Te Chen C, Li LY, et al. Myeloid cell leukemia-1 in- et al. Structure at 2.5-Å resolution of chemically synthesized human versely correlates with glycogen synthase kinase-3β activity and associates with A.M. Ali et al. / Computational and Structural Biotechnology Journal 17 (2019) 263–281 281

poor prognosis in human breast cancer. Cancer Res 2007;67:4564–71. https://doi. [125] Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activa- org/10.1158/0008-5472.CAN-06-1788. tion of the p53 pathway by small-molecule antagonists of MDM2. Science 2004; [109] Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, et al. Estrogen re- 303. https://doi.org/10.1126/science.1092472 80-. 844–8. ceptors: how do they signal and what are their targets. Physiol Rev 2007;87: [126] Schulte TW, Neckers LM. The benzoquinone ansamycin 17-allylamino-17- 905–31. https://doi.org/10.1152/physrev.00026.2006. demethoxygeldanamycin binds to HSP90 and shares important biologic activities [110] Clarke M. Meta-analyses of adjuvant therapies for women with early breast cancer: with geldanamycin. Cancer Chemother Pharmacol 1998;42:273–9. https://doi. the early breast Cancer Trialists' collaborative group overview. Ann Oncol 2006;17: org/10.1007/s002800050817. x59–62. https://doi.org/10.1093/annonc/mdl238. [127] Bird GH, Mazzola E, Opoku-Nsiah K, Lammert MA, Godes M, Neuberg DS, et al. Bio- [111] Abe O, Abe R, Enomoto K, Kikuchi K, Koyama H, Masuda H, et al. Effects of chemo- physical determinants for cellular uptake of hydrocarbon-stapled peptide helices. therapy and hormonal therapy for early breast cancer on recurrence and 15-year Nat Chem Biol 2016. https://doi.org/10.1038/nchembio.2153. survival: an overview of the randomised trials. Lancet 2005;365:1687–717. [128] Lalatsa A, Schatzlein AG, Uchegbu IF. Strategies to deliver peptide drugs to the https://doi.org/10.1016/S0140-6736(05)66544-0. brain. Mol Pharm 2014;11:1081–93. https://doi.org/10.1021/mp400680d. [112] Tan YS, Lane DP, Verma CS. Stapled peptide design: principles and roles of compu- [129] Melmed S, Popovic V, Bidlingmaier M, Mercado M, van der Lely AJ, Biermasz N, tation. Drug Discov Today 2016;21:1642–53. https://doi.org/10.1016/j.drudis.2016. et al. Safety and efficacy of Oral octreotide in acromegaly: results of a multicenter 06.012. phase III trial. J Clin Endocrinol Metab 2015;100:1699–708. https://doi.org/10. [113] Cornillie SP, Bruno BJ, Lim CS, Cheatham TE. Computational modeling of stapled 1210/jc.2014-4113. peptides toward a treatment strategy for CML and broader implications in the de- [130] Brayden DJ, Alonso M-J. Oral delivery of peptides: opportunities and issues for sign of lengthy peptide therapeutics. J Phys Chem B 2018;122:3864–75. https://doi. translation. Adv Drug Deliv Rev 2016;106:193–5. https://doi.org/10.1016/J.ADDR. org/10.1021/acs.jpcb.8b01014. 2016.10.005. [114] Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. [131] Wallbrecher R, Chène P, Ruetz S, Stachyra T, Vorherr T, Brock R. A critical assess- UCSF chimera - a visualization system for exploratory research and analysis. J ment of the synthesis and biological activity of p53/human double minute 2- Comput Chem 2004;25:1605–12. https://doi.org/10.1002/jcc.20084. stapled peptide inhibitors. Br J Pharmacol 2017;174:2613–22. https://doi.org/10. [115] Cornell WD, Cieplak P, Bayly CI, Gould IR, Kenneth M, Merz J, et al. A second gener- 1111/bph.13834. ation force field for the simulation of proteins, nucleic acids, and organic molecules. [132] Okamoto T, Zobel K, Fedorova A, Quan C, Yang H, Fairbrother WJ, et al. Stabilizing J Am Chem Soc 1995;117:5179–97. https://doi.org/10.1021/ja00124a002. the pro-apoptotic BimBH3 helix (BimSAHB) does not necessarily enhance affinity [116] Hopkins CW, Le Grand S, Walker RC, Roitberg AE. Long-time-step molecular dy- or biological activity. ACS Chem Biol 2013;8:297–302. https://doi.org/10.1021/ namics through hydrogen mass repartitioning. J Chem Theory Comput 2015;11: cb3005403. 1864–74. https://doi.org/10.1021/ct5010406. [133] Moellering RE, Cornejo M, Davis TN, Del Bianco C, Aster JC, Blacklow SC, et al. Direct [117] Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, et al. An inhibition of the NOTCH transcription factor complex. Nature 2009;462:182–8. inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005; https://doi.org/10.1038/nature08543. 435:677–81. https://doi.org/10.1038/nature03579. [134] Weng Z, Zhao Q. Utilizing ELISA to monitor protein-protein interaction. Protein- [118] Vu BT, Vassilev L. Small-molecule inhibitors of the p53-MDM2 interaction. Curr Top protein interact. Methods appl2nd ed. ; 2015. https://doi.org/10.1007/978-1- Microbiol Immunol 2011;348:151–72. https://doi.org/10.1007/82_2010_110. 4939-2425-7_21 p. 341–52. [119] Maloney A, Workman P. HSP90 as a new therapeutic target for cancer therapy: the [135] Mattheyses AL, Marcus AI. Förster resonance energy transfer (FRET) microscopy for story unfolds. Expert Opin Biol Ther 2002;2:3–24. https://doi.org/10.1517/ monitoring biomolecular interactions. Protein-protein interact. Methods appl2nd 14712598.2.1.3. ed. ; 2015. https://doi.org/10.1007/978-1-4939-2425-7_20 p. 329–39. [120] Neckers L, Workman P. Hsp90 molecular chaperone inhibitors: are we there yet? [136] Bacart J, Corbel C, Jockers R, Bach S, Couturier C. The BRET technology and its appli- Clin Cancer Res 2012;18:64–76. https://doi.org/10.1158/1078-0432.CCR-11-1000. cation to screening assays. Biotechnol J 2008;3:311–24. https://doi.org/10.1002/ [121] Trudel S, Zhi HL, Rauw J, Tiedemann RE, Xiao YW, Stewart AK. Preclinical studies of biot.200700222. the pan-Bcl inhibitor obatoclax (GX015-070) in multiple myeloma. Blood 2007; [137] Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, et al. 109:5430–8. https://doi.org/10.1182/blood-2006-10-047951. Direct observation of individual endogenous protein complexes in situ by proxim- [122] Nguyen M, Marcellus RC, Roulston A, Watson M, Serfass L, Murthy Madiraju SR, ity ligation. Nat Methods 2006;3:995–1000. https://doi.org/10.1038/nmeth947. et al. Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes [138] Bernal F, Wade M, Godes M, Davis TN, Whitehead DG, Kung AL, et al. A stapled p53 MCL-1-mediated resistance to apoptosis. Proc Natl Acad Sci 2007;104:19512–7. Helix overcomes HDMX-mediated suppression of p53. Cancer Cell 2010;18: https://doi.org/10.1073/pnas.0709443104. 411–22. https://doi.org/10.1016/j.ccr.2010.10.024. [123] Paik PK, Rudin CM, Pietanza MC, Brown A, Rizvi NA, Takebe N, et al. A phase II study [139] Herce HD, Deng W, Helma J, Leonhardt H, Cardoso MC. Visualization and targeted of obatoclax mesylate, a Bcl-2 antagonist, plus topotecan in relapsed small cell lung disruption of protein interactions in living cells. Nat Commun 2013;4. https://doi. cancer. Lung Cancer 2011;74:481–5. https://doi.org/10.1016/j.lungcan.2011.05. org/10.1038/ncomms3660. 005. [140] Li Y, Rodewald LW, Hoppmann C, Wong ET. A versatile platform to analyze low af- [124] Billard C. Design of novel BH3 mimetics for the treatment of chronic lymphocytic finity and transient protein- protein interactions in living cells in real time, vol. 9; leukemia. Leukemia 2012;26:2032–8. https://doi.org/10.1038/leu.2012.88. 2014; 1946–58. https://doi.org/10.1016/j.celrep.2014.10.058.A.